MECHANISTIC CHARACTERIZATION OF THE ATP HYDROLYSIS ACTIVITY
OF ESCHERICHIA COLI LON PROTEASE USING KINETIC TECHNIQUES
by
DIANA VINEYARD
Submitted in partial fulfillment of the requirements for the
degree of Doctor of Philosophy
Thesis Adviser: Dr. Irene Lee
Department of Chemistry
CASE WESTERN RESERVE UNIVERSITY
January, 2007
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
Diana Vineyard
______________________________________________________
candidate for the Ph.D. degree *.
Robert D. Salomon
(signed)_______________________________________________
(chair of the committee)
Mary Barkley
________________________________________________
John Mieyal
________________________________________________
Irene Lee
________________________________________________
Lawrence M. Sayre
________________________________________________
________________________________________________
11-09-06
(date) _______________________
*We also certify that written approval has been obtained for any
proprietary material contained therein.
TABLE OF CONTENTS
TITLE PAGE…………………………………………………………………………........i
COMMITTEE SIGN OFF SHEET…………………………………………………...insert
TABLE CONTENTS……………………………………………………………………..ii
LIST OF FIGURES………………………………………………………………………vi
LIST OF TABLES………………………………………………………………………..xi
LIST OF SCHEMES……………………………………………………………………xiii
LIST OF ABBREVIATIONS…………………………………………………………...xiv
ACKNOWLEDGEMENTS……………………………………………………………xviii
ABSTRACT…………………………………………………………………………….xix
CHAPTER 1: INTRODUCTION TO LON PROTEASE………………………………..1
CHAPTER 2: DEVELOPMENT OF STANDARD ASSAY FOR STEADY-STATE
ATPase ACTIVITY AND IDENTIFICATION OF CONFORMATIONAL
CHANGE ASSOCIATED WITH NUCLEOTIDE BINDING………………….19
2.1 INTRODUCTION………………………………………………………………......20
2.2 MATERIALS & METHODS……………………………………………………….26
2.2.1 Penefsky Columns………………………………………………………...26
2.2.2 Malachite Green NTPase Assays………………………………………….26
2.2.3 Radiolabeled NTPase Assays…………………………………………..…28
2.2.4 Tryptic Digestions…………………………………………………………29
2.3 RESULTS & DISCUSSION……………………………………………………….30
ii
CHAPTER 3: ASSESSING THE TIMING OF MECHANISTIC EVENTS IN
ESCHERICHIA COLI LON PROTEASE THROUGH CHARACTERIZATION
OF ITS PRE-STEADY-STATE ATPase ACTIVITY…………………………...47
3.1 INTRODUCTION………..…………………………………………………………48
3.2 MATERIALS & METHODS……………………………………………………….52
3.2.1 Chemical Quench ATPase Activity Assays……………………………….52
3.2.2 Filter Binding Assay………………………………………………………54
3.2.3 Bradford Assay……………………………………………………………54
3.2.4 Pulse Chase ATPase Activity Assays……………………………………..55
3.3 RESULTS & DISCUSSION………………………………………………………..56
CHAPTER 4: SINGLE TURNOVER EXPERIMENTS ISOLATE THE ATPase
ACTIVITY AT THE HIGH-AFFINITY SITES IN ESCHERICHIA COLI LON
PROTEASE……………………………………………………………………...85
4.1 INTRODUCTION…………………………………………………………………..86
4.2 MATERIALS & METHODS……………………………………………………….91
4.2.1 Double Filter Binding Assay…………………………………………………91
4.2.2 Single Turnover ATPase Assays……………………………………………..92
4.2.3 Chemical Quench ATPase Assays……………………………………………93
4.2.4 Tryptic Digestions…………………………………………………………….94
iii
CHAPTER 5: (MDCC)-LABELED PHOSPHATE BINDING PROTEIN IS USED TO
PROBE PHOSPHATE RELEASE IN THE ATPase MECHANISM OF
ESCHERICHIA COLI LON PROTEASE……………………………………...118
5.1 INTRODUCTION…………………………………………………………………119
5.2 MATERIALS & METHODS……………………………………………………...123
5.2.1 Cloning and Expression of Phosphate Binding Protein (PBP)……………...123
5.2.2 Fermenting PBP……………………………………………………………..123
5.2.3 Purification and MDCC-Labeling of PBP…….…………………………….125
5.2.4 Checking the Activity of MDCC-PBP…………………...………………….126
5.2.5 Steady-State ATPase Assays Using MDCC-PBP…………………………..126
5.2.6 Stopped Flow Experiments using MDCC-PBP…………………………….127
5.3 RESULTS & DISCUSSION………………………………………………………128
CHAPTER 6: THE FLUORESCENT NUCLEOTIDE ANALOGUE (MANT-ATP) IS
USED TO PROBE THE KINETIC MECHANISM OF ESCHERICHIA COLI
LON PROTEASE………………………………………………………………142
6.1 INTRODUCTION…………………………………………………………………143
6.2 MATERIALS & METHODS……………………………………………………...146
6.2.1 Steady-State MANT-ATPase Assays using Malachite Green………………146
6.2.2 Steady-State MANT-ATPase Assays using MDCC-PBP…………………..147
6.2.3 Peptidase Methods…………………………………………………………..148
6.2.4 Tryptic Digestions…………………………………………………………...149
6.2.5 MANT-ATP Binding Time Courses by Fluorescent Stopped Flow….……..150
iv
6.2.6 MANT-ADP Release Time Courses by Fluorescent Stopped Flow……...…150
6.3 RESULTS & DISCUSSION………………………………………………………152
CHAPTER 7: CONCLUSIONS AND FUTURE DIRECTIONS: A REVISED
KINETIC MODEL FOR THE ATP HYDROLYSIS ACTIVITY IN
ESCHERICHIA COLI LON PROTEASE……………………………………...178
7.1 INTRODUCTION…………………………………………………………………179
7.2 MATERIALS & METHODS……………………………………………………...188
7.2.1 Four Syringe Rapid Quench Assay………………………………………….188
7.2.2 Double Mixing Stopped Flow Assay………………………………………..189
7.2.3 Cloning Y461W Lon Mutant………………………………………………..190
7.2.4 Stopped Flow Intrinsic Fluorescence Assays……………………………….191
7.2.5 Crystal Screening……………………………………………………………191
7.3 RESULTS & DISCUSSION………………………………………………………193
APPENDIX A – List of Kinetic Equations……………...……………………………...219
APPENDIX B – First Author Publications……………………………………………..223
REFERENCES…………………………………………………………………………224
v
LIST OF FIGURES
CHAPTER 1
Figure 1.1 Depiction of normal and Lon deficient mitochondrion……………………….3
Figure 1.2 a) Domain organization of E. coli Lon b) Crystal structure of protease
domain of S679A E. coli Lon c) Partial crystal structure of α-domain of E. coli Lon…..5
Figure 1.3 Structures of nonhydrolyzable ATP analogs………………………………….9
Figure 1.4 Model for Lon activity proposed by Goldberg………………………………..9
Figure 1.5 Explanation of continuous fluorescent peptidase assay developed by Lee and
Berdis. Included is a structure of S1 peptide……………………………………………11
Figure 1.6 Structure of S2 peptide substrate, the non-fluorescent analog of S1………...13
Figure 1.7 Peptidase activity of E. coli Lon protease (125 nM Lon, 1 mM ATP, varying
50 – 500 µM S3 peptide)………………………………………………………………...15
CHAPTER 2
Figure 2.1 Amino acid sequence alignment of the AAA+ ATPases: Lon, HslU, and ClpA
from E. coli………………………………………………………………………………21
Figure 2.2 Crystal structure of HslU depicting the closed conformational change in the
presence of ATP and ADP……………………………………………………………….23
Figure 2.3 Structures of the purine and pyrimidine bases found in the nucleotide triphosphates………………………………………………………………………………..25
Figure 2.4 Steady-state NTPase activity of E. coli Lon protease monitored by the
malachite green assay……………………………………………………………………32
Figure 2.5 Steady-state NTPase activity of E. coli Lon protease monitored by the
radioactive assay…………………………………………………………………………35
Figure 2.6 Limited tryptic digestion of Lon in the presence of ATP, ADP, AMPPNP,
CTP, GTP, and UTP……………………………………………………………………..38
Figure 2.7 Fragmentation of Lon resulting from limited tryptic digestion and the relative
positions of the fragments compared to the intact Lon monomer……………………….40
vi
Figure 2.8 Model proposed for the different enzyme forms in Lon that couple ATP
binding and hydrolysis in activating peptide hydrolysis…………………………………45
CHAPTER 3
Figure 3.1 Base catalyzed hydrolysis of benzylidenemalanitrile at room temperature as
monitored using the rapid quench (KinTek Corp)……………………………………….59
Figure 3.2 Bar graph showing the effectiveness of the various denaturing quenches in
inactivating Lon protease within 5 ms………………………………………………...…61
Figure 3.3 Pre-steady-state S2 stimulated ATPase activity of Lon protease quenched
with formic acid, HCl, and EDTA……………………………………………………….64
Figure 3.4 Pre-steady-state S2 stimulated ATPase activity of E. coli Lon protease
showing the fit of the burst equation……………………………………………………..66
Figure 3.5 Pre-steady-state ATPase activity of E. coli Lon protease a) intrinsic, S2
stimulated, and casein stimulated b) zooms in on the burst region…………………….67
Figure 3.6 Pre-steady-state S2 stimulated ATPase activity at increasing concentrations of
ATP 5 – 200 µM………………………………………………………………………....71
Figure 3.7 Analysis of pre-steady-state rate constants obtained from the fit of Figure 3.6
a) steady-state rate constants vs. [ATP] b) Burst amplitudes vs. [ATP] c) Burst rates
vs. [ATP]……………………………………………………………………………..71-72
Figure 3.8 Filter binding assay a) Bio-Rad Dot-Blot assembly b) Representative
phosphorimaged nitrocellulose membrane………………………………………………75
Figure 3.9 Filter binding assay data. pmol of 32P nucleotide vs. µL Lon monomer……79
Figure 3.10 Pre-steady-state ATPase activity of E. coli Lon protease. Comparison of
pulse-chase and acid quench experiment in the presence and absence of S2 peptide…...78
Figure 3.11 Proposed model for Lon ATP hydrolysis activity………………………….81
Figure 3.12 Comparison of pre-steady-state ATP hydrolysis and S3 cleavage by E. coli
Lon protease……………………………………………………………………………...84
CHAPTER 4
vii
Figure 4.1 Kd titration of the high- and low-affinity ATPase sites using the double filter
binding assay……………………………………………………………………………..98
Figure 4.2 Single turnover experiment to detect ATP hydrolysis at the high-affinity site
on the rapid quench…………………………………………………………………….100
Figure 4.3 Bench top pre-steady-state time courses of ATPase activity of E. coli Lon
under single turnover conditions in the presence and absence of S2 peptide…………..102
Figure 4.4 Bench top assay to measure high-affinity ATPase activity as the ATP:Lon
ratio increases………………………………………………………………………...…104
Figure 4.5 Bench top assay to measure high-affinity ATPase activity when saturating
amounts of ATP, ADP, and AMPPNP are spiked in following the first half life of the
reaction at the high-affinity sites………………………………………………………..106
Figure 4.6 Limited tyrptic digestion of E. coli Lon under single turnover conditions
compared to pseudo-first order conditions……………………………………………..109
Figure 4.7 Pre-steady-state acid quenched time course of S2 stimulated ATP hydrolysis
at only the low-affinity sites…………………………………………………………....112
Figure 4.8 Pre-steady-state acid quenched time courses of intrinsic ATP hydrolysis in the
presence of varying amounts of ADP…………………………………………………..114
Figure 4.9 IC50 plot of steady-state rate constants associated with ATP hydrolysis at
increasing concentrations of ADP……………………………………………………...116
CHAPTER 5
Figure 5.1 Structure of the MDCC-thiol adduct……………………………………….120
Figure 5.2 SDS-PAGE visualized by Coomassie Brilliant Blue of pHF019 PBP induction
study…………………………………………………………………………………….129
Figure 5.3 Depiction of the phosphate “mop” system………………………………....131
Figure 5.4 SDS-PAGE visualized by Coomassie Brilliant Blue showing that PBP and
PNPase are not separated by size exclusion chromatography………………………….131
Figure 5.5 Emission scan of 2 µM MDCC-PBP from a) pHF019 or b) original cell
strain…………………………………………………………………………………….132
Figure 5.6 Calibration curve of 250 µM MDCC-PBP in Lon reaction buffer…………134
viii
Figure 5.7 Comparison of steady-state ATPase activity of E. coli Lon in the presence and
absence of S2 peptide as determined by the MDCC-PBP assay or the radioactive ATPase
assay…………………………………………………………………………………….135
Figure 5.8 Fluorescent stopped-flow time courses of Lon casein stimulated ATPase
activity with increasing amounts of MDCC-PBP………………………………………137
Figure 5.9 Calibration curve of 25 µM MDCC-PBP using fluorescent stopped flow…137
Figure 5.10 a) Detection of ATPase activity of E. coli Lon protease in the absence and
presence of casein at varying concentrations of ATP b) zooms in on first 0.5 s………138
Figure 5.11 Detection of ATPase activity of E. coli Lon in the presence of increasing
amounts of Pi “mop”……………………………………………………………………140
CHAPTER 6
Figure 6.1 Chemical structure of MANT-ATP………………………………………...145
Figure 6.2 Emission scans of MANT-ATP showing the increase in fluorescence when
bound to E. coli Lon…………………………………………………………………….153
Figure 6.3 Steady-state MANT-ATPase activity of Lon as determined using the
malachite green colorimetric assay……………………………………………………..154
Figure 6.4 Steady-state MANT-ATPase activivty of Lon as determined using the
MDCC-PBP fluorescent assay………………………………………………………….156
Figure 6.5 MANT-ATP supports S3 peptide cleavage comparably to ATP…………..158
Figure 6.6 SDS-PAGE visualized by Coomassie brilliant blue shows MANT-ATP and
MANT-AMPPNP induces the same conformational change as ATP………………….160
Figure 6.7 Determining the Kd for the low-affinity ATPase sites in E. coli Lon using
MANT-ATP…………………………………………………………………………….162
Figure 6.8 a) Representative time course of MANT-ATP binding to Lon b) The two
observed rate constants extracted from binding time courses plotted vs. [ATP]……….164
Figure 6.9 Representative time course of MANT-dATP binding to E. coli Lon………167
Figure 6.10 Representative time course of MANT-ADP release from E. coli Lon in a
single mixing stopped flow experiment………………………………………………...169
ix
Figure 6.11 Representative time course of MANT-dADP release from E. coli Lon in a
single mixing stopped flow experiment………………………………………………...171
Figure 6.12 Representative time course of MANT-ADP release from E. coli Lon in a
double mixing experiment, isolating the low-affinity site……………………………...174
Figure 6.13 Representative time courses of MANT-ADP release following the hydrolysis
of MANT-ATP for a) 35 s or b) 50 s in double mixing experiments, isolating the lowaffinity site……………………………………………………………………………...175
CHAPTER 7
Figure 7.1 Collective fit of the acid-quench ATPase data using FitSim………………195
Figure 7.2 Simulation of the expected time courses of “F” form hydrolysis………….197
Figure 7.3 Depiction of the 4-syringe quench experiment to monitor the first round of
ATPase activity by the “F” form……………………………………………………….199
Figure 7.4 Four syringe rapid quench time course of ATP hydrolysis………………...199
Figure 7.5 Representative time courses of MANT-ADP release from the “F” form in
double mixing experiments in the presence and absence of S2 peptide………………..203
Figure 7.6 Crystal structure of HslU showing the four distinct conformational states of
the enzyme depending on nucleotide di- or tri-phosphate being bound………………..207
Figure 7.7 a) SDS-PAGE visualized by Coomassie brilliant blue shows Y461W E. coli
Lon mutant displays same adenine specific conformational change as wild type b)
Fluorescent peptidase assay shows that Y461W mutant displays comparable peptidase
activity to wild type……………………………………………………………..……...208
Figure 7.8 Stopped flow experiments monitor changes in intrinsic tryptophan
fluorescence in Y461W versus wild type Lon………………………………………….210
Figure 7.9 SDS-PAGE visualized by silver staining shows pure wild type E. coli Lon
and smaller ∆-N E. coli Lon mutant……………………………………………………212
Figure 7.10 Fluorescent peptidase assay shows the ∆-N E. coli Lon mutant displays
comparable peptidase activity to wild type Lon………………………………………..212
Figure 7.11 Picture of the salt crystals from Hampton Research Crystal Screen 1
condition 18…………………………………………………………………………….216
x
LIST OF TABLES
CHAPTER 1
Table 1.1 Comparison of Substrate Specificity Constants for Various Peptide and Protein
Substrates of Lon Protease………………………………………………………….……13
Table 1.2 Summary of Steady-State Kinetic Parameters of ATP – and AMPPNPDependent Cleavage of S3 by E. coli Lon……………………………………………….15
CHAPTER 2
Table 2.1 Steady-State Kinetic Parameters of NTP Hydrolysis by E. coli Lon as
Determined by the Malachite Green Assay……………………………………………...33
Table 2.2 Steady-State Kinetic Parameters of NTP Hydrolysis by E. coli Lon as
Determined by the Radioactive NTPase Assay……………………………………….....36
Table 2.3 Identification of the Tryptic Digestion Sites in Lon………………………….40
CHAPTER 3
Table 3.1 Summary of Kinetic Parameters Obtained from Fitting the Pre-Steady-State
ATPase (intrinsic, S2 stimulated, casein stimulated)…………………………………….69
Table 3.2 Summary of the Pre-Steady-State Kinetic Parameters Associated with S2
Stimulated APT Hydrolysis by Lon Protease……………………………………………73
CHAPTER 4
Table 4.1 Rate Constants Associated with High-Affinity Site ATPase Activity…...…107
Table 4.2 Rate Constants Associated with ADP Inhibition of Intrinsic ATP
Hydrolysis………………………………………………………………………………115
CHAPTER 5
Table 5.1 Summary of ATPase Steady-State Kinetic Parameters from Comparison of
MDCC-PBP and [α-32P]ATP Assays…………………………………………………..135
xi
CHAPTER 6
Table 6.1 Comparison of the Steady-State Parameters Associated with the Hydrolysis of
ATP and MANT-ATP…………………………………………………………………..154
Table 6.2 MANT-ATP and ATP Steady-State Kinetic Parameters Associated with ATP
Hydrolysis………………………………………………………………………………156
Table 6.3 MANT-ATP and ATP Steady-State Kinetic Parameters Associated with S3
Cleavage………………………………………………………………………………...158
Table 6.4 Rate Constants Associated with Nucleotide Binding as Determined by
Fluorescent Stopped Flow………………………………………………………………166
Table 6.5 Comparison of the Rate Constants Associated with MANT-ATP Binding and
MANT-ADP Release with MANT-dATP……………………………………………...167
Table 6.6 Summary of MANT-ADP Release Rate Constants from Single Mixing
Stopped Flow Experiments…………………………………………………………….171
CHAPTER 7
Table 7.1 Comparison of Experimentally Obtained Rate Constants with those
Determined from the Collective Fit of the Data to the Kinetic Mechanism in Scheme
7.3.....................................................................................................................................194
Table 7.2 Summary of MANT-Nu Off Rates from “Blocked” or Free Lon in Double
Mixing Experiments…………………………………………………………………….203
Table 7.3 Summary of Crystal Screens Set up in van den Akker Lab………………....214
Table 7.4 Summary of Crystal Screens Performed with Inhibitors……………………216
xii
LIST OF SCHEMES
CHAPTER 1
Scheme 1.1 Proposed Ser-Lys dyad catalysis of amide bond cleavage by Lon Protease...7
Scheme 1.2 Propose kinetic mechanism for E. coli Lon protease based upon steady-state
kinetic studies…………………………………………………………………………….16
CHAPTER 3
Scheme 3.1 Schematic of KinTek Corporation rapid quench………………………...…50
Scheme 3.2 Depiction of base catalyzed hydrolysis of benzylidenemalanitrile………...57
Scheme 3.3 Minimal kinetic mechanism for ATP hydrolysis by Lon protease………...61
Scheme 3.4 Illustration of the difference between an acid quench and pulse chase rapid
quench experiment……………………………………………………………………….78
CHAPTER 4
Scheme 4.1 Predicted nucleotide bound enzyme forms with various ATP
concentrations……………………………………………………………………………89
CHAPTER 7
Scheme 7.1 Revised catalytic mechanism for intrinsic ATPase activity………………180
Scheme 7.2 Revised catalytic mechanism for S2 stimulated ATPase activity………...184
Scheme 7.3 Mechanism used for the collective fit of the kinetic data using the program
FitSim…………………………………………………………………………………...194
xiii
LIST OF ABBREVIATIONS
λ N protein
a λ phage protein that allows E. coli RNA polymerase to transcribe
through termination signals in the early operons of the phage
AAA+
ATPases Associated with Various Cellular Activities
Abz
Anthranilamide
ADP
Adenosine Diphosphate
AM
Ammonium Molybdate
AMPPCP
Adenylyl (β,γ-methylene) Diphosphonate – a “nonhydrolysable” ATP
analogue
AMPPNP
Adenyl 5-Imidotriphosphate – a “nonhydrolyzable” ATP analogue
ATP
Adenosine Triphosphate
ATP-γ-S
Adeosine 5’-O-(3-Thiotriphosphate)
BSA
Bovine Serum Albumin
Bz
Benzoic Acid Amide
CTP
Cytidine Triphosphate
DNA
Deoxyribonucleic Acid
DTT
Dithiothreitol
E. coli
Escherichia coli a gram negative bacteria
EDTA
Ethylenediaminetetraacetic Acid
FRET
Fluorescence Resonance Energy Transfer
GDP
Guanosine Diphosphate
GTP
Guanosine Triphosphate
HEPES
N-2-Hydroxyethylpiperazine-N’-Ethanesulphonic Acid
IPTG
Isopropyl-β-D-Thiogalactopyranoside
xiv
KATP
The concentration of ATP required to reach ½ the maximal burst
amplitude
kburst
Burst rate constant for ATP hydrolysis
kcat
Vmax / [E]
kcat/Km
Substrate specificity constant
Kd
Dissociation constant
Km
Michaelis Constant = [substrate] required to reach ½ Vmax
kobs
ν/[E]
kon,MANT-Nu
The rate constant associated with binding of nucleotide which is
dependent on [Nu]
kon,2
The rate constant associated with binding of nucleotide which is not
dependent on [Nu]
koff, MANT-Nu
The rate constant associated with the release of nucleotide also shown as
koff,1 and koff,2
kss,ATP
Steady-state rate constant for ATP hydrolysis obtained from rapid quench
time courses
kss,S3
Steady-state rate constant for S3 hydrolysis = kcat at saturating [S3]
KOAc
Potassium Acetate
KPi
Potassium Phosphate
LB
Luria-Bertani Medium
MANT
2’(3’)-O-(N-Methylanthraniloyl)
MDCC
7-Diethylamino-3-[[[(2-Maleimidyl)Ethyl]Amino]Carbonyl]Coumarin
MDCC-PBP A197C mutant of PBP labeled with MDCC
7-MEG
7-Methylguanosine a substrate of PNPase
MG
Malachite Green
Mg(OAc)2
Magnesium Acetate
xv
n
Hill Coeficient
NDP
Nucleotide Diphosphate
NO2
Nitro
NTP
Nucleotide Triphosphate
NTPase
Nucleotide Triphosphate Hydrolysis
Nu
Nucleotide
P11
Phosphocellulose resin used for cation exchange
Pi
Inorganic Phosphate
PBP
Phosphate Binding Protein from E. coli
Pd1
The hydrolyzed product of S2 containing the last five amino acids from
the carboxyl terminal of S2
Pd2
The hydrolyzed product of S2 containing the last five amino acids from
the amino terminal of S2
PEI-cellulose Polyethyleneimine-cellulose
PNPase
Purine Nucleoside Phosphorylase
PMT
Photomultiplier Tube on the stopped flow instrument
S1
Also known as the FRETN 89-98 peptide in our previous study, contains
amino acid residues 89-98 of the λ N protein
[Y(NO2)RGITCSGRQK(Abz)
S2
A nonfluorescent analogue of S1 that is cleaved by Lon identically as S1
[YRGITCSGRQKBz]
S3
A mixed peptide substrate containing 10% S1 and 90% S2 designed to
overcome the inner filter effect observed in S1
SBTI
Soybean Trypsin Inhibitor
SDS-PAGE
Sodium Dodecylsulfate Poly-Acrylamide Gel Electrophoresis
SSD
Sensor and Substrate Discrimination
xvi
Tris
Tris(hydroxylmethyl)aminomethane
UTP
Uridine Triphosphate
ν
Rate (of Peptide or Nucleotide Hydrolysis)
Vmax
Maximal Rate (of Peptide or Nucleotide Hyrolysis)
xvii
ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Irene Lee, for her guidance and support during my
time at Case Western. I was incredibly lucky to have such an approachable,
understanding, and patient mentor as a boss. The camaraderie of my fellow labmates
Hilary Frase, Jennifer Fishovitz, Dr. Xuemei Zhang, and especially my best friend Jessica
Ward, who was instrumental in my progress towards graduation, kept me driven and I
appreciate all of their assistance and encouragement. I would like to thank Dr. Focco van
den Akker and Dr. Pius Padayatti for their assistance in teaching me how to perform
protein crystallization screens. Thank you also to Dr. Anthony Berdis for being available
for helpful scientific discussion. Finally a big thank you to my family and my fiancé,
Tom, for all of their support; without them I would never have made it this far.
xviii
Mechanistic Characterization of the ATP Hydrolysis Activity of Escherichia coli Lon
Protease Using Kinetic Techniques
Abstract
by
DIANA VINEYARD
Lon is an oligomeric serine protease whose proteolytic activity is activated by
ATP hydrolysis. Because the ATPase and protease activities of Lon are inevitably
intertwined, a clear understanding of the role of ATP hydrolysis in the enzyme is
necessary in elucidating the reaction mechanism of Lon and related ATP-dependent
proteases. Lon displays an intrinsic ATPase activity which is stimulated by the presence
of peptide or protein substrate. Other purine and pyrimidine triphosphates activate Lon
protease activity however ATP is the best activator of peptide cleavage. Additionally
peptide stimulates ATP hydrolysis to the highest degree compared to the other nucleotide
triphosphates. Collectively these results indicate that ATP is the preferred substrate of
Lon. Although each monomeric subunit has an identical sequence, Lon contains two
types of ATPase sites that have differing affinities for ATP as well as drastically differing
rates of ATP hydrolysis. In this dissertation I primarily utilize pre-steady-state kinetic
techniques to monitor the rate constants associated with ATP binding and hydrolysis as
well as ADP and inorganic phosphate release from Lon. As stated above both a highand low-affinity ATPase site exist in Lon which display approximately 20-fold
differences in affinity and approximately 1000 fold differences in their rate of ATP
xix
hydrolysis. Therefore the roles of the two ATPase sites in Lon catalysis were probed in
more detail in order to determine their individual effect on peptidase activity and if they
were communicating with one another. On the basis that neither the rate of ATP binding,
hydrolysis, nor product release was affected by the presence of peptide substrate but the
steady-state turnover number is stimulated by peptide, a unique enzyme form of Lon was
proposed to exist. This enzyme form named “F”, is implicated to be generated
subsequent to the first round of ATP hydrolysis and catalytically hydrolyzes both peptide
and ATP. Taken together, my work demonstrated that the ATPase activity of Lon
behaves like a molecular motor which couples the binding and hydrolysis of ATP in a
manner similar to those found in other AAA+ proteins to drive protein degradation.
xx
CHAPTER 1
INTRODUCTION TO LON PROTEASE
1
Lon protease, also known as protease La, is an ATP-dependent serine protease
functioning to degrade damaged and certain short-lived regulatory proteins in the cell
independently of the ubiquitin pathway (for reviews, (1-3)). Escherichia coli (E. coli)
cells deficient in the lon gene (lon- mutants) display phenotypes such as increased
sensitivity to UV light, decreased levels of intracellular proteolysis, and the cells display
an elongated appearance, thus the name Lon was assigned to the protein (4, 5). Lon is
found ubiquitously in nature, distributed throughout the cytosol in prokaryotes, and is
localized to the mitochondria in eukaryotic cells (6). In eukaryotes, Lon maintains the
proper function of the mitochondria by ensuring correct folding and assembly of nascent
polypeptides, and by preserving the integrity of the mitochondrial genome (Figure 1.1)
(6-12). Mitochondrial dysfunction has been implicated in various degenerative disorders
including aging, Alzheimer’s disease, and cancer (13-15). Therefore, elucidating the
precise function and mechanism of Lon protease will provide insight in the design of
strategies to prevent mitochondrial related diseases. Furthermore, recent studies targeting
the proteins necessary for virulence in pathogenic bacteria including Brucella abortus
(16), Pseudomonas syringae (17), and Salmonella enterica have identified Lon (18-20).
As pathogenic forms of these bacteria are responsible for diseases ranging from mild
gastroenteritis to undulant and typhoid fever and the numbers of antibiotic resistant
strains have increased, Lon could prove to be an important therapeutic target in
pathogenic bacteria. Since the E. coli homologue shares 42% sequence identity and
functional homology with the eukaryotic forms, as well as greater than 99% sequence
identity with S. Typhimurium Lon, it can be used as a model system to study the
mechanism of Lon (21, 22).
2
normal
mitochondrion
Lon deficient
mitochondrion
Figure 1.1 – Depiction of normal and Lon deficient mitochondrion. Adapted from
Suzuki, CK et al (1994) (6).
3
In E. coli Lon, the molecular weight of each monomeric subunit in Lon is ~89
kDa and the monomers consist of four domains (23, 24). From the N- to C-terminus
these domains are the N-terminal domain, the ATPase domain, the substrate sensor and
discriminatory domain (SSD), and the protease domain (Figure 1.2a). Like other ATPdependent proteases found in E. coli including FtsH, ClpAP, ClpXP, and HslUV, Lon
belongs to the AAA+ (ATPases associated with various cellular activities) superfamily of
proteins (25, 26). This family is defined by a characteristic nucleotide binding domain
whose conserved regions includes the Walker A and B motifs (27-29). Unlike the other
ATP-dependent proteases, the ATPase and protease domains in Lon are located within
each monomeric subunit (23, 30). No structure of intact wild type Lon is available
however portions of the enzyme have been crystallized (31-33). Figure 1.2b shows the
hexameric structure of E. coli Lon protease (P) domain where the six monomers are
arranged in a dome-shaped hexamer containing a central pore (32). Figure 1.2c shows a
side view of the hexameric protease domain with one “ATPase” domain modeled on the
protease domain through a linker region (31). The “ATPase” domain is written in
quotation marks because the structure does not include the conserved Walker A and B
nucleotide binding motifs. Rather, Botos and colleagues collectively name the ATPase
and SSD domains as the “ATPase” domain, and the pictured structure is more
representative of the SSD domain. Site directed mutagenesis studies of Lon have
identified the serine residue at position 679 as the active site serine responsible for amide
bond cleavage (4). This residue was mutated to an alanine in the pictured crystal
structure. Because of the proximity of the Lys722 side chain to the mutated Ala679 in the
4
a
H3N+
N-terminal
domain
ATPase
domain
ba
sensor and substrate
discriminator domain
protease
domain
COO-
Peptidase
domain
18Å
bc
“ATPase”
domain
Peptidase
domain
32Ǻ
Figure 1.2 – (a) Domain organization of E. coli Lon in the primary sequence. (b)
Structure of E. coli Lon protease (“P”) domain. The monomers form a dome-shaped
hexamer with a central pore. (c) Side view of the protease domain with the “ATPase”
domain is modeled above the protease domain through a linker region. Adapted from
Botos, et al, 2004 (32).
5
crystal structure and the lack of other catalytic side chains, the authors proposed that Lon
employs a Ser-Lys dyad in the active site for catalysis. The mechanism for catalysis is
most likely similar to that of the Thr-N terminal amino group dyad used by the
proteasome (Scheme 1.1) (34).
Although Lon requires ATPase activity to degrade peptide or protein substrate,
the ATPase activity is not fully coupled to the peptidase activity as the enzyme
hydrolyzes ATP in the absence of peptide substrate (35). The segregation of the ATPase
activity is also demonstrated by the ability of the proteolytically inactive S679A mutant
to hydrolyze ATP (35, 36). As stated above, the primary sequence of Lon shares the
nucleotide binding cassette known as the Walker A sequence which is conserved in
AAA+ enzyme family (27-29). Walker A consists of a phosphate binding loop (P-loop)
that has been shown to be essential for the binding of the nucleotide tri-phosphates. The
P-loop is comprised of a glycine-rich sequence followed by a conserved lysine and a
serine or threonine residue. When the conserved lysine residue was mutated to alanine
(K362A), the resulting dramatic decrease in the mutant’s peptidase activity confirms the
mechanistic link between the two activities (36). Because the ATPase and protease
activities of Lon are inevitably intertwined, a clear understanding of the role of ATP
hydrolysis in the enzyme is necessary.
It has been proposed that Lon functions as an oligomer the size of which has not
been definitely determined in solution, however the oligomerization of Lon is
independent of ATP binding or hydrolysis (37). Lon exhibits a basal level of “intrinsic”
ATPase activity which is stimulated in the presence peptide or protein substrate. One
product of ATP hydrolysis, ADP, binds more tightly to Lon than ATP and inhibits both
6
Peptide or
protein
substrate
NH
NH
Nucleophilic
attack
Lys
O
679
Ser
P1'
HN
NH2
O-
P1
O
P1
722
Tetrahedral
intermediate
HO
Lys722
P1'
HN
H
+
NH2
O
679
Ser
P1'
O
Upstream
Product
Formation of
acyl-enzyme
H2N
NH
P1
O
Regeneration of
Active Site
NH
O
NH
OH
Downstream
Product
Lys722
P1
OH
+
H2N
O
OH
679
Ser
NH2
O
H
Acyl-enzyme
hydrolysis
O
P1
O
Acyl-enzyme
intermediate
H
Ser679
Lys722
Scheme 1.1 – Proposed Ser-Lys dyad catalysis of amide bond cleavage by Lon Protease.
7
the ATPase and protease activity of the enzyme (38, 39). The products of the protease
activity of the enzyme are typically five to ten amino acids in length (1, 38, 39).
Although it is known that the coordination between the protease and ATPase activities is
necessary for maximal protease activity, the specifics of how the two activities are
coordinated with one another is unknown. Nonhydrolyzable analogues of ATP such as
AMPPNP (Figure 1.3) were shown to support the cleavage of protein substrates albeit not
as effectively as ATP (1, 40). This suggests that the binding and hydrolysis of ATP have
different effects on the activation of the peptidase activity of Lon. Early kinetic data
suggested that the connection between ATP hydrolysis and peptide binding was
attributed to an energy dependent step used to present peptide or protein substrate to the
active site (3, 35). Figure 1.4 shows a mechanism for ATP-dependent protein breakdown
by the protease Lon proposed previously (1). This model was based on findings
including the proposed existence of an allosteric site that binds protein substrate and the
ability of protein substrate to facilitate the release of ADP from Lon (3). The model
indicates an “active” and “inactive” form of Lon, which is dictated by the presence of the
nucleotide tri- or di-phosphate respectively. It predicts that peptide cleavage occurs
before ATP hydrolysis, and the catalytic turnover of peptide relies on the substrates
ability to promote ADP/ATP exchange. Given the available information about Lon, this
model served as a reasonable interpretation of the data. However, it does not specifically
address the coordination of the ATPase and protease activities.
Limitations in the study of Lon were partially due to the lack of a peptide
substrate that interacts with the enzyme in a comparable manner to a protein substrate.
Small synthetic peptides had been used to study the protease activity of Lon previously
8
NH2
N
O
O
-
P
P
O-
N
O
O
H
N
O
O-
P
N
O
O
O-
H
H
OH
OH
H
AMPPNP
N
H
NH2
N
O
O-
P
O-
P
N
O
O
H2
C
O
O-
P
N
O
N
O
O-
H
H
OH
OH
H
AMPPCP
H
NH2
N
O
O-
P
S
O
P
O-
N
O
O
O
P
O
N
O
O-
H
N
H
H
OH
OH
H
H
ATP- γ−S
Figure 1.3 – Structures of non-hydrolyzable ATP analogs. Although ATP-γ-S is very
slowly hydrolysable it is often referred to as a non-hydrolyzable analog (41-43).
inactive
protease
(E•ADP)
protein
substrate
peptide
products
substrate
hydrolysis
Pi + Mg 2+
allosteric
activation by
substrates
ADP
ATP + Mg2+
active
protease
(E•Mg-ATP)
Figure 1.4 – It was proposed that the interaction of the substrate with an allosteric site in
Lon leads to the release of ADP and the partial activation of the protease activity. The
binding and hydrolysis of Mg-ATP further facilitates the protease activity, and as long as
the allosteric site is occupied ATP/ADP exchange will occur. Adapted from Goldberg
(1992) (44).
9
but their hydrolysis required only ATP binding, whereas the degradation of protein
substrates also required ATP hydrolysis (3, 45-49). Illustrating this point was the finding
that nonhydrolyzable ATP analogs including AMPPCP, and AMPPNP as well as the
slowly hydrolysable analog ATPγS (Figure 1.3) supported the degradation of tetrapeptide
substrates of chymotrypsin by Lon equivalent to ATP, but did not support the degradation
of protein substrates (1). One peptide substrate Glt-Ala-Ala-Phe-MNA even inhibited
ATP hydrolysis (49). Furthermore, only protein and not the small peptide substrates
stimulated ATP hydrolysis. This phenomenon was explained by the hypothesis that
structural features on the protein were the recognition elements for the peptidase site, and
that ATP hydrolysis was necessary in Lon to both unfold the substrate protein and make
the scissile peptide bond accessible (1, 3, 44). This hypothesis reconciled the energy
requirement from ATP hydrolysis in the Lon-mediated degradation of structured proteins
including casein (45) and CCdA (50). However, the ATPase activity of Lon protease is
also stimulated by the λ N protein (46). The λ N protein is a bacteriophage protein which
is a physiological substrate of E. coli Lon protease that does not adopt any secondary
structure in the absence of RNA (51, 52). Thus the hypothesis did not account for the
stimulation of and requirement for ATP hydrolysis in the degradation of λ N protein,
because it would predict little or no ATPase stimulation because no substrate unfolding is
necessary (46). In addition protein substrates contain many cleavage sites and therefore
make it difficult to directly correlate the binding and hydrolysis of ATP to a specific
10
H3N-Y(NO2)-RGITCSGRQ-K(Abz)-COO
NO 2
HN
OH
λN 89-98
(S1)
H 2N
-
relative fluorescence
+
1 10
5
8 10
4
6 10
4
4 10
4
2 10
4
O
0
0
ATP, Lon
+
H3N-Y(NO2)-RGITC
50
100
150
200
250
300
tim e (s)
SGRQ-K(Abz)-COO-
Figure 1.5 – Explanation of the continuous fluorescent peptidase assay developed by Lee
and Berdis. S1 peptide (residues 89-98 of λ N protein) contains a fluorescent donor,
anthranilamide (Abz) on the carboxy terminus and a fluorescence quencher, 3nitrotyrosine (Y-(NO2)) on the amino terminus. The S1 peptide is cleaved by Lon in the
presence of ATP between the cysteine (C) and serine (S) residues resulting in a
separation of the fluorescent donor and quencher and a concomitant increase in
fluorescence. The assay is ideal for mechanistic characterization of Lon because the S1
peptide displays kinetic parameters comparable to the λ N protein.
11
cleavage event. To address this issue, Lee and Berdis (53) developed a ten amino acid
synthetic peptide mimic (S1) of the λ N protein (residues 89-98) containing a single
cleavage site and no secondary structure in order to perform detailed mechanistic
characterization on the coordination of the peptidase and ATPase activities in Lon
protease. A fluorescence donor, anthranilamide (Abz), was engineered on the carboxy
terminus and the fluorescence quencher, 3-nitrotyrosine (Y-(NO2)), on the amino
terminus (Figure 1.5). The minimally fluorescent peptide mimic is cleaved by Lon in the
presence of ATP between the cysteine (C) and serine (S) residues resulting in the
separation of the fluorescent donor and quencher, and consequently an increase in
fluorescence corresponding to a cleavage event. Therefore, the peptidase activity of Lon
can be measured in real time (excitation 320 nm, emission 420 nm) using fluorescent
spectroscopy and kinetic parameters can be obtained by calibrating the fluorescent signal
with trypsin (Figure 1.5). The length of the peptide was intentionally made longer than
the tetrapeptides used in the past because the products of Lon protease activity are
typically between five to ten amino acids in length. As the cleavage site in S1 peptide
lies in the middle of the sequence the resulting peptide products are five amino acids in
length which corresponds to typical size of protein degradation products. S1 also
stimulated ATP hydrolysis comparably to a protein substrate. One complication resulting
from the fluorescent peptide was the occurrence of the technical limitation known as the
inner filter effect (54). At concentrations of S1 peptide > 150 µM, the fluorescence due
to the cleavage of the peptide deviates from the linear relationship between the signal and
the amount of peptide hydrolyzed because nitrotyrosine absorbs anthranilamide
12
cleavage site
+
H3N Y RGITCSGRQ K(Bz)
CO2-
HN
OH
O
Figure 1.6 – Depiction of S2 peptide substrate, the non-fluorescent analog of S1 peptide.
Table 1.1: Comparison of Substrate Specificity Constants* (kcat/Km) for Various Peptide
and Protein Substrates of Lon Protease
λN protein
kcat/Km
x 104 (M-1 s-1)
7.7a
S3 (λN 89-98)
8.8b
Casein
1.3a
Glt-Ala-Ala-Phe-MNA
0.0027c
Substrate
*
The larger specificity constants correspond to better substrates.
The kinetic constants were obtained from reference (46)
b
The kinetic constants were obtained from reference (55)
c
The kinetic constants were obtained from reference (49)
a
13
fluorescence through an inter-molecular mechanism rather that an intra-molecular
mechanism. However, the linear range of the substrate was expanded by incorporating
the use of a non-fluorescent analog of S1, known as S2 peptide. As shown in Figure 1.6,
S2 peptide substitutes the nonfluorescent benzoic acid moiety at the carboxy terminus for
the anthranilamide and tyrosine for the 3-nitrotyrosine at the amino terminus. When a
mixture of 10% S1 and 90% S2 (termed S3) is used, the linear range of fluorescence was
expanded to at least ten times that of S1 alone. S1, S2, and S3 are all degraded with
identical kinetics by E. coli Lon protease (40). Table 1.1 shows a comparison of the
substrate specificity constants of S3 peptide with other protein and peptide substrates. As
one would expect for a peptide “mimic”, the specificity constant of S3 is nearly identical
to that of λ N protein.
ATP-mediated S3 cleavage is shown in Figure 1.7. The velocity plots of S3
cleavage display sigmoidal kinetics with a resulting n value between 1.6 and 2, which
suggests a degree of cooperativity between the subunits during catalysis. The kinetic
constants for ATP- and AMPPNP-mediated S3-cleavage are summarized in Table 1.2.
AMPPNP-mediated catalysis is approximately seven fold slower than ATP-mediated
catalysis despite comparable Km (Table 1.2) values for the nucleotides and similar Ks
(concentration of nucleotide needed to reach ½ maximal velocity) values (40). Using S3
peptide, Thomas-Wohlever and Lee (40) performed an in depth steady-state mechanistic
characterization of E. coli Lon protease. They used a combination of ADP and product
inhibition studies as well as a comparison of ATP and AMPPNP activated S3 cleavage to
gauge the effects of ATP binding and hydrolysis to expand upon the existing model for
14
8
k
obs
-1
(s )
6
4
2
0
0
100
200
300
400
500
[S3 peptide], µM
Figure 1.7 – 1 mM ATP was incubated with 125 nM E. coli Lon protease at
concentrations of peptide increasing from 50 – 500 µM. Peptide cleavage was monitored
on a Fluorimeter (excitation 320 nm, emission 420 nm), the time courses transformed
from signal per time to [S3] cleaved per time using trypsin to generate a linear calibration
curve. The kobs values were calculated at the various concentrations of peptide and
plotted versus peptide concentration in the above figure. The data were fit using the Hill
equation to generate the kinetic parameters which are summarized in Table 1.2.
Table 1.2: Summary of Steady-State Kinetic Parameters of ATP- and AMPPNPDependent Cleavage of S3 by E. coli Lon
ATPa
kcat
(s-1)
9.0 + 0.5
Km
(µM)
102 + 30
1.6
AMPPNPb
1.0 + 0.1
77 + 7
1.6
Nucleotide
a
b
N
values obtained from reference (55)
values obtained from reference (40)
15
3
2
E + ADP
E:ADP
E:ATP
1
a
E + ATP
1'
E
S3
E
b
F
ATP
Pd1 + Pd2
S3
ATP
c
ATP
F
7'
S3
E
ATP
2'
Pi
E*
ADP
S3*
6'
S3
3'
S3 E*
F Pd1 + Pd2
ADP
S3*
ATP
E* S3*
ADP
S3
4'
5'
E*
S3*
Scheme 1.2 – Proposed kinetic mechanism for E. coli Lon protease based upon steadystate kinetic studies. Adapted from Thomas-Wohlever and Lee (40).
16
Lon catalysis. Scheme 1.2 depicts the proposed kinetic mechanism derived from this
study (40). Steps 1-3 delineate the intrinsic hydrolysis of ATP in the absence of S3
peptide substrate. When S3 is present, the hydrolysis of ATP facilitates the translocation
of S3 to the peptidase site, and at saturating concentrations of S3, binding of S3 to the
allosteric site promotes ADP/ATP exchange (steps 1’-5’). Upon subsequent binding of a
second molecule of ATP, the translocated peptide, S3*, is cleaved yielding the peptide
products Pd1 and Pd2 (step 6’). This step is accompanied by an enzyme isomerization
step (E*→F) to yield a post-catalytic form of Lon whose conversion back to the precatalytic enzyme form is required for turnover, but the intricacies of this process were not
determined. In contrast to the mechanism proposed by Goldberg shown in Figure 1.4,
this model predicts ATP hydrolysis to occur prior to peptide cleavage.
The goal of the studies presented in this dissertation is to verify the validity of the
kinetic mechanism proposed in Scheme 1.2 by identifying the microscopic rate constants
associated with the ATPase activity. In order to accomplish this goal, a standard steadystate kinetic assay for NTPase activity was optimized and a variety of pre-steady-state
kinetic techniques were utilized to measure rate constants for ATP binding and hydrolysis
as well as product release from Lon. As pre-steady-state kinetic analyses allow for the
examination of enzymatic activity on a time scale of a single enzyme turnover, rate
constants corresponding to individual steps in the reaction pathway were measured. For
example rapid chemical quench experiments allowed for the examination of the rate of
the first turnover of ATP hydrolysis. These experiments indicated the Lon displays halfsite reactivity with regard to the ATPase activity. The proposed half-site reactivity was
dissected in more detail using single turnover experiments. Furthermore, stopped flow
17
experiments were employed to monitor the rate of ATP binding and product release from
Lon. When the rate constants obtained from the pre-steady-state studies were compiled
into a revised mechanism for the ATPase activity of Lon it was evident that the enzyme
functions uniquely compared to other proteins in the AAA+ family. The revised ATPase
mechanism serves as a scaffold for establishing the various facets of the enzyme’s
peptidase activity which is being investigated in detail by Jessica Patterson-Ward.
18
CHAPTER 2
DEVELOPMENT OF A STANDARD ASSAY FOR STEADY-STATE ATPase
ACTIVITY AND IDENTIFICATION OF CONFORMATIONAL CHANGE
ASSOCIATED WITH NUCLEOTIDE BINDING
19
Introduction
Although the microscopic details are unknown, the ATPase activity of Lon was
suggested to facilitate the protease activity of the enzyme (45). Lon displays an intrinsic
ATPase activity in the absence of any peptide or protein substrate (48, 55). The function
of the intrinsic ATP hydrolysis is not understood, however the ATPase and peptidase
activities were proposed to be coupled because the ATP hydrolysis activity is stimulated
in the presence of S2 peptide (40, 53) or protein substrates (1, 35). The development of a
working model for Lon’s ATPase activity would aid in the comprehensive understanding
of the mechanism of Lon as well as the coordination of the ATPase and peptidase
activities of the enzyme. Lon belongs to the AAA+ (ATPases associated with diverse
cellular activities) class of ATPases which display chaperone-like activity related to the
assembly, operation, and disassembly of protein complexes (25). Other ATP-dependent
proteases such as ClpAP, ClpXP, and HslUV are also AAA+ enzymes (26). The AAA+
enzymes share two consensus motifs associated with nucleotide binding which were first
identified by Walker and colleagues in 1982 (28). The first motif, named Walker A
(GXXXXGKT/S; X can be varied) is also known as the phosphate binding loop in
proteins when the threonine residue is replaced with cysteine, and is used as a hallmark
for a nucleotide binding protein. Walker B (ZZZZD; Z is a hydrophobic residue) is the
second sequence motif identified by Walker and colleagues and is always on the cterminal side relative to Walker A in the primary sequence (27). Figure 2.1 depicts a
sequence alignment of the ATPase domain of E. coli Lon with ClpA, and HslU (25).
The heterosubunit ATP-dependent protease HslUV is the bacterial homologue of the
proteasome (56). In this enzyme the oligomeric ATPase subunit (HslU) is separate
20
Walker A
Lon
HslU
ClpA
Walker B
351 ILCLVGPPGVGKTSLGQSIAKAT 417 NPLFLLDEIDKMS-----52 NILMIGPTGVGKTEIARRLAKLA 250 HGIVFIDEIDKICKRGESS
490 SFLFAGPTGVGKTEVTVQLSKAL 558 HAVLLLDEIEKA-------
Sensor 1
Sensor 2
452 FSDHYLEVDYDLSDVMFVATS---NIPAPLLDRME 539 AGVRGLEREISKLCRKAVKQL
299 TDHILFIASGAFQIAKPSD----LIPELQG--------RLP 390 IGARRLHTVLERLMEEISYDA
596 FRNVVLVMTTNAGVRETERK[21]TPEFRN---RLD 699 MGARPMARVIQDNLKKPLANE
Figure 2.1 – Amino acid sequence alignment of the AAA+ ATPase: Lon, HslU, and
ClpA from E. coli. The conserved Walker A and B motifs are highlighted along with
conserved arginine residues in the sensor motifs which are proposed to interact with the
phosphate backbone of the nucleotide triphosphate. Adapted from Ogura, et. al. (57),
Neuwald et. al. (25), and Yoshida and Amano (29).
21
from the oligomeric protease subunit (HslV). The two subunits come together to form
the functional enzyme which functions to degrade proteins in the presence of ATP. Both
the HslU and HslV subunits contain central pores that align in the HslUV complex. The
HslU ATPase subunit regulates the unfolding and translocation of polypeptide substrates
which were proposed to be threaded through the central cavity for degradation (58-60).
HslU is the only AAA+ protease to be crystallized with different nucleotides (61). By
comparing the crystal structures of HslU not bound to any nucleotide with HslU
complexed with the nucleotide di- and tri-phosphate molecules the authors were able to
identify a conformational change in the enzyme associated with adenine nucleotide
binding. As shown in Figure 2.2, rotation of the α-helical domain in HslU results in four
distinct states of the protein where the “empty” is the most “open” conformation and the
“ADP” bound is the most “closed” conformation. In fact, conserved arginine residues in
AAA+ ATPases have been implicated in interacting with the phosphate residues of the
nucleotide and “sensing” differences between the di- and tri- phosphates (57). Analysis
of the crystal structure of HslVU suggested that the nucleotide-dependent conformational
change facilitated a protein unfolding-coupled translocation mechanism where the
substrate protein is threaded through the central pore of the enzyme complex in order to
be degraded (62). Although no structure has been solved for intact Lon, a structural
model of the α−sub-domain and protease domain in Lon was superimposable on the
HslUV structure (32). Therefore, our lab utilizes HslUV as a structural model in
studying the ATP-dependent peptidase reaction of Lon and hypothesize that Lon also
adopts a similar nucleotide-dependent conformational change similar to HslU.
22
Figure 2.2 – There are four distinct HslU conformational states. They are the
ADP/dADP (silver), ATP (cyan), SO4 (magenta), and empty (orange) states. The αhelical domain rotates as a rigid body along a single axis near Box VII’ marked by the X.
The rotation resulting from the various states are listed with respect to the ADP/dADP
conformational state. Adapted from Wang, et al. (61).
23
This chapter describes the development of a reliable assay to probe the steadystate ATPase activity of Lon and the utilization of a low resolution structural probe to
identify the proposed adenine-specific conformational change. Lon is known to
hydrolyze ATP as well as CTP, GTP, and UTP (Figure 2.3) in the presence and absence
of casein (35), but a quantitative summary of the kinetic parameters has not been
reported. Thus, the steady-state NTPase activity of Lon in the presence and absence of
our model peptide substrate S2 (non-fluorescent analog of S1) was examined using the
optimized assay conditions (55). The quantitative kinetic parameters obtained from these
studies allowed us to test the validity of the minimal kinetic model for Lon (40) discussed
in Chapter 1. We further probed the structural consequence of the various nucleotides on
Lon using limiting amouts of trypsin to digest Lon into polypeptide fragments.
Comparing the resulting digestion patterns in the presence and absence of nucleotide
serves as a low resolution structural probe for a nucleotide-dependent conformational
change. The combination of the kinetic and structural data allowed us to evaluate the
functional role of a proposed adenine-specific conformational change in Lon as well as to
quantitatively asses the relationship between the peptidase and ATPase activities.
24
NH2
N
O
N
N
H
N
NH
N
H
N
Adenine
N
NH2
Guanine
O
NH2
NH
N
N
H
Cytosine
O
N
H
O
Uracil
Figure 2.3 – Structures of the purine and pyrmidine bases found in the nucleotide triphosphates.
25
Materials and Methods
Materials. Nucleotides were purchased from ICN Biomedical (CTP lot 2077F, GTP lot
9311C, UTP lot 6688F) or Sigma (ATP lot A-7699). The [α-32P]ATP, [α-32P]CTP, [α32
P]GTP, and [α-32P]UTP were purchased from Perkin-Elmer Life Science. Tris buffer,
PEI-cellulose TLC plates, SBTI, TPCK-treated trypsin, ammonium molybdate, sodium
citrate, malachite green oxalate salt were purchased from Fisher.
General Methods. Peptide synthesis and protein purification procedures were performed
as described previously (40). All enzyme concentrations were reported as Lon monomer
concentrations. All reagents were reported as final concentrations. Unless otherwise
stated all experiments were performed at 37 º C.
Penefsky Column. ~2.6 g of Sephadex G50 beads were boiled in 65 mL of 50 mM Tris
pH 8, 75 mM KOAc, 2 mM DTT, 0.01% Tween 20, and 20% glycerol for 15 min. The
presence of the Tween 20 detergent ensures the recovery of most of the protein from the
column. The Sepahdex G50 suspension was then chilled at 4 °C. The slurry was then
loaded into a 1 mL or 3 mL syringe which had been plugged with a polystyrene disk.
The syringe columns were spun in test tubes to collect the flow thru at 1000 rpm for 2
min to pack the column. The columns were switched to new test tubes and the protein
loaded (~100 µL protein/1 mL column or ~300 µL protein/3 mL column). The columns
were again spun to exchange the buffer at 1000 rpm for 2 min. The eluted protein was
tested with malachite green to ensure that the phosphate had been eliminated. If not, the
procedure was repeated until the phosphate contamination was gone.
Malachite Green NTPase Assays. Steady-state velocity data for ATP, CTP, GTP and
UTP were collected using a modified colorimetric assay to detect the release of inorganic
26
phosphate (Pi) from the nucleotide tri-phosphates as the gamma phosphate was cleaved
by Lon protease (63, 64). Solutions containing 0.045% (w/v) malachite green oxalate
(MG) in deionized water, 4.2% (w/v) ammonium molybdate (AM) in 4 N HCl, 2% Triton
X-100 in deionized water, and 34% (w/v) sodium citrate•2H2O in deionized water were
prepared. Prior to each NTPase assay a 3:1 mixture of MG:AM was made, stirred for at
least 20 min, and filtered through 0.4 µM filter paper. The Triton X-100 solution was
then added to the MG/AM solution in the amount of 100 µL per 5 mL of 3:1 MG/AM
solution. A solution of NaHPO4 and NaH2PO4 (pH 8.1) was used as a calibration
standard.
For the NTPase measurements, a 210 µL reaction mixture containing 50 mM Tris
buffer pH 8.1, 5 mM magnesium acetate (Mg(OAc)2), 2 mM DTT, various
concentrations of Lon protease (125 nM for ATP) as well as 500 µM peptide substrate
(S2, the nonfluorescent analogue of S3) for the peptide stimulated NTPase assays was
used. For all assays the NTPase reaction was initiated with the addition of the nucleotide
to the reaction mixture, and all reactions were performed at least in triplicate. At 8 time
points (from 0 min to 15 min) a 25 µL aliquot was thoroughly mixed with 400 µL of
MG/AM/Triton X solution. After 30 s, 50 µL of 34% sodium citrate was added for color
development. The absorbance was then recorded at 660 nm on a UV-vis
spectrophotometer for each of the time points. The amount of Pi formed at each time
point was determined by comparing the absorbance of the sample to a Pi calibration
curve. Initial velocities were determined from plots of the amount of Pi released versus
time. The kinetic parameters were determined by fitting the averaged rate data with the
27
Michaelis Menten equation (eq 1) using the nonlinear regression program KaleidaGraph
(Synergy) version 3.6.
k obs =
k obs , max [ Nu ]
K m + [ Nu ]
(1)
where kobs is the observed rate constant, kobs,max is the maximal rate constant, [Nu] is the
concentration of nucleotide, and Km is the Michaelis-Menten constant equal to the
concentration of Nu required to reach one half the maximal rate constant.
Radiolabeled NTPase Assays. Steady state velocity data were collected as described
previously (63) and all reactions were performed at least in triplicate. Briefly, for the
NTPase measurements, each reaction mixture (50 µL) contained 50 mM Tris-HCl (pH
8.1), 5 mM Mg(OAc)2, 2 mM DTT, and 150 nM Lon monomer for ATP or UTP while
CTP and GTP required 600 nM Lon monomer. For the peptide-stimulated NTPase
reactions, 500 µM peptide substrate (S2) was added to each reaction mixture. All
reactions were initiated with the addition of [α-32P]NTP and subsequently 5 µL aliquots
were quenched in 10 µL of 0.5 N formic acid at seven time points (from 0 to 12 min). A
3 µL aliquot (ATPase) or 2 µL aliquot (CTPase, GTPase, and UTPase) of the reaction
was spotted directly onto a PEI-cellulose TLC plate (10 cm X 20 cm) and developed in
0.3 M potassium phosphate buffer pH 3.4. Radiolabeled NDP nucleotide was then
quantified using the Packard Cyclone storage phosphor screen Phosphor imager
purchased from Perkin Elmer Life Science. To compensate for slight variations in
spotting volume the concentration of NDP product obtained at each time point was
corrected for using an internal reference as shown in equation 2.
28


NDPdlu
 × [ NTP ]
[ NDP ] = 
 NTPdlu + NDPdlu 
(2)
where [NDP] is the concentration of the nucleotide diphosphate, [NTP], is the
concentration of the nucleotide triphosphate, NDPdlu are the density light units
corresponding to the nucleotide diphosphate, and NTPdlu are the density light units
corresponding to the nucleotide triphosphate. Initial velocities were determined from
plots of the amount of NDP produced over time. The kinetic parameters were determined
by fitting the kobs data with eq 1 using the nonlinear regression program KaleidaGraph
(Synergy) version 3.6.
Tryptic Digestions. Tryptic digestion reactions containing 1.5 µM Lon, 50 mM Tris-HCl
(pH 8.1), 5 mM Mg(OAc)2, 2 mM DTT, with or without 800 µM S2 peptide, and either 1
mM ATP, ADP, or AMPPNP, 2 mM CTP or GTP, or 3 mM UTP were started by the
addition of 1/50 (w/w) TPCK (N-p-tosyl-L-phenylalanine chloromethyl ketone)-treated
trypsin with respect to Lon. At 0, 15 and 30 min, a 3 µL reaction aliquot was quenched
in 3 µg soybean trypsin inhibitor (SBTI) and 3 µL 5x loading dye followed by boiling.
The quenched reactions were then fractionated on a SDS-PAGE gel and visualized with
Coomassie brilliant blue.
29
Results and Discussion
Colorometric Assay for Inorganic Phosphate (Pi) Monitors Lon ATP Hydrolysis Activity.
As the ATP-dependent Lon protease displays an intrinsic ATPase activity which is
stimulated in the presence of peptide or protein substrate, it can be characterized as an
ATPase (35). The homology of the ATPase domain of Lon with other AAA+ proteins
allows for the comparison of mechanistic similarities shared by the enzymes including
ATP-dependent conformational change, and the need for ATP hydrolysis to induce work
(61). Therefore, the first step in the characterization of the ATPase activity of Lon is to
establish a reliable assay to monitor the steady-state kinetics of the enzyme. The steadystate NTPase activity was measured using both a radioactive and colorimetric
discontinuous assay (55). The colorimetric assay utilizes malachite green dye which
undergoes a change in absorbance at 660 nm when a phosphomolybdate-malachite green
complex is formed (65) presumably following the hydrolysis of the gamma phosphate of
the NTPases by Lon. Because the presence of phosphate salt stabilizes E. coli Lon and
prevents protein precipitation from occurring, the enzyme is typically stored in a buffer
containing 75 mM KPi. Prior to using a colorimetric assay which detects inorganic
phosphate, the residual phosphate in the enzyme storage buffer was reduced using the
rapid size exclusion chromatography method known as a Penefsky column as described
in the Materials and Methods. Although the background phosphate from the enzyme was
reduced using size exclusion chromatography, it in combination with the Pi contamination
from other reagents as well as the nucleotides themselves made it difficult to accurately
monitor NTP hydrolysis. The residual phosphate in the reagents increased the signal to
noise ratio such that, the malachite green colorimetric assay was not an optimal method
30
for detecting ATPase activity in the Lon system. However, because it is an accepted
method for monitoring ATPase activity malachite green was used to characterize Lon
NTPase activity (66-71). Although the data is relatively scattered, the hyperbolic plots in
Figure 2.4 demonstrates that the hydrolysis of ATP, CTP, GTP and UTP by Lon display
Michaelis-Menten kinetics. The maximal rate constant kcat, NTP and Michaelis constant,
Km, were then obtained by fitting the data shown in Figure 2.4 with equation 1. The
resulting kinetic parameters are summarized in Table 2.1. Table 2.1 shows that the error
among the Km values for intrinsic NTPase and peptide stimulated ATPase and GTPase
activity varies considerably. The intrinsic kcat values show that the trend for how fast Lon
hydrolyzes the NTPs is UTP > ATP > CTP > GTP. A increased catalytic efficiency is
indicated by higher kcat/Km value indicates a preferred substrate because it selects for
substrates with faster kcat values and lower Km values which typically indicate better
substrate binding (72). As shown by the kcat/Km values in Table 2.1, ATP is clearly the
preferred substrate for Lon protease.
Radiolabeled Assay for ADP Monitors Lon ATP Hydroysis Activity. However, because
the malachite green assay uses a large amount of reagent and is not ideal for the Lon
system for the reasons explained above, we also optimized a radioactive assay to monitor
Lon’s NTPase activity in order to confirm the data. To this end radio-nucleotides with
32
P at the alpha phosphate position were purchased so that the production of the
[α−32P]NDP could be monitored as Lon cleaves [α−32P]NTP. The alpha labeled
phosphate was used in preference to the gamma phosphate because side by side
comparison revealed that the [γ-32P]NTP had a slightly higher product contamination as
opposed to [α-32P]NTP which increased the signal to noise ratio in the assay. The
31
b
0.8
0.2
0.6
0.15
obs
-1
(s )
0.25
k
k
obs
-1
(s )
a
1
0.4
0.1
0.2
0.05
0
0
0
200
400
600
800
0
1000
200
[ATP], µM
400
600
800
1000
[CTP], µM
c
d
0.014
1.2
0.012
1
0.01
-1
(s )
0.008
obs
0.006
0.6
k
k
obs
-1
(s )
0.8
0.4
0.004
0.2
0.002
0
0
200
400
600
800
0
1000
0
1000
2000
3000
4000
5000
[UTP], µM
[GTP], µM
Figure 2.4 – Steady-State NTPase activity of E. coli Lon Protease Monitored by
Malachite Green Assay. (a) ATP hydrolysis in the presence (■) and absence (●) of 800
µM S2 peptide at ATP concentrations including 50, 75, 100, 250, and 500 µM and 1 mM.
(b) CTP hydrolysis in the absence (●) of S2 peptide at CTP concentrations including 50,
75, 100, 250, and 500 µM and 1 mM. (c) GTP hydrolysis in the presence (■) and
absence (●) of S2 800 µM peptide at GTP concentrations including 50, 75, 100, 175, 250,
and 500 µM and 1 mM. (d) UTP hydrolysis in the absence (●) of S2 peptide at UTP
concentrations including 100, 200, and 500 µM as well as 1, 2, 3, and 5 mM. The rate
constants reported are average values of different trials.
32
Table 2.1: Steady – State Kinetic Parameters of NTP Hydrolysis by E. coli Lon as
Determined by the Malachite Green Assay
Intrinsic
S2-stimulated
Km,NTP
(µM)
60 + 11
kcat / Km
(x 103 M-1s-1)
3.2
kcat, NTP
(s-1)
1.2 + 0.03
Km,NTP
(µM)
230 + 16
kcat / Km
(x 103 M-1s-1)
5.2
NTPase
enhancement
ATP
kcat, NTP
(s-1)
0.19 + 0.01
CTP
0.28 + 0.02
150 + 25
1.9
ND*
ND*
ND*
ND*
GTP
0.007 + 0.001
39 + 18
0.18
0.01 + 0.001
74 + 14
0.14
1.4
UTP
1.6 + 0.03
1700 + 740
1.2
ND*
ND*
ND*
ND*
Nucleotide
*
ND – values not determined due to technical limitations of the assay
33
6.3
nucleotide di- and tri-phosphates were separated using strong anion exchange thin layer
chromatography and the amounts quantified using Phosphor imaging. Figure 2.5 shows
the steady-steady NTPase activity of E. coli Lon protease as monitored by the radioactive
assay (55). The hydrolysis of all the nucleotides displays Michaelis–Menten kinetics
which agrees with the hyperbolic dependence of the observed rate constants (kobs) on the
concentration of nucleotide. The solid black lines in Figure 2.5 show the fit of the data
with equation 1 and the kinetic parameters summarized in Table 2.2. Because the kinetic
parameters for ATPase activity as monitored by both the malachite green assay (Table
2.1, ATP) and the radioactive assay (Table 2.2, ATP) were on the same order of
magnitude the radioactive assay was pursued as the standard assay. Had a more
extensive study been pursued using the malachite green assay to probe CTP, GTP, and
UTP hydrolysis which included multiple trials and higher quality reagents, I would
expect the kinetic parameters between the two assays to be more comparable. Again the
malachite green assay requires more reagents, has more steps, and in general is not ideal
for measuring ATPase activity in Lon because of the high background phosphate (Pi)
contamination in our system. As discerned in Figure 2.5, the NTPase activity of Lon is
moderately stimulated in the presence of saturating amounts of S2 peptide for all
nucleotides tested. The NTPase enhancement is reported in Table 2.2 as the ratio of the
peptide stimulated kcat over the intrinsic kcat. The Km values of the nucleotides are
comparable in the presence and absence of peptide. The difference in the nucleotides lies
more in their observed rate constants associated with hydrolysis by Lon. The trend of kcat
values for intrinsic NTP hydrolysis is UTP > ATP > CTP > GTP and ATP ~ UTP > CTP
> GTP in the presence of S2 peptide. The kcat/Km value is highest for ATP in the
34
a
b
1.4
0.25
1.2
0.2
0.15
-1
(s )
1
k
obs
k
obs
-1
(s )
0.8
0.6
0.1
0.4
0.05
0.2
0
0
0
200
400
600
800
1000
0
1200
200
400
[ATP], µM
c
0.12
1000
1200
1
0.1
-1
(s )
0.8
obs
0.08
0.6
k
k
obs
-1
800
d
1.2
0.14
(s )
600
[CTP], µM
0.06
0.4
0.04
0.2
0.02
0
0
0
100
200
300
400
500
600
0
500
1000
1500
2000
[UTP], µM
[GTP], µM
Figure 2.5 - Steady-State NTPase activity of E. coli Lon Protease Monitored by a
Radioactive Assay. (a) [α32P]ATP hydrolysis in the presence (■) and absence (●) of 800
µM S2 peptide at ATP concentrations including 25, 50, 100, 250, and 500 µM and 1 mM.
(b) [α32P]CTP hydrolysis in the presence (■) and absence (●) of S2 peptide at CTP
concentrations including 25, 50, 100, 250, and 500 µM and 1 mM. (c) [α32P]GTP
hydrolysis in the presence (■) and absence (●) of S2 800 µM peptide at GTP
concentrations including 25, 50, 100, 250, and 500 µM. (d) [α32P]UTP hydrolysis in the
presence (■) and absence (●) of S2 peptide at UTP concentrations including 50, 100, 250,
and 600 µM as well as 1, and 2 mM. All kinetic assays were performed at least in
triplicate and the observed rate constants reported are average values of these different
trials.
35
Table 2.2: Steady – State Kinetic Parameters of NTP Hydrolysis by E. coli Lon as
Determined by the Radioactive NTPase Assay
Intrinsic
S2-stimulated
Km,NTP
(µM)
46 + 6
kcat / Km
(x 103 M-1s-1)
5.7
kcat, NTP
(s-1)
1.4 + 0.05
Km,NTP
(µM)
82 + 10
kcat / Km
(x 103 M-1s-1)
17
NTPase
enhancement
ATP
kcat, NTP
(s-1)
0.26 + 0.01
CTP
0.13 + 0.02
17 + 13
7.6
0.25 + 0.01
49 + 8
5.1
2
GTP
0.09 + 0.01
65 + 31
1.4
0.15 + 0.01
41 + 11
3.7
1.7
UTP
0.49 + 0.02
91 + 16
5.4
1.1 + 0.06
130 + 37
8.5
2.2
Nucleotide
36
5.4
presence of peptide suggesting that ATP is the best activator of peptide hydrolysis and
confirming the malachite green data that suggested ATP was the preferred substrate.
Limited Tryptic Digestion Probes Structural Changes in the AAA+ Motif in Lon. In order
to correlate the steady-state kinetic NTPase data with structural changes in the AAA+
motif in Lon (25, 26, 31), we utilized tryptic digestion which is a low resolution structural
probe. Limited proteolysis experiments such as tryptic digestion can indicate the domain
organization of a protein if the proteolysis fragments are sequenced and can also suggest
conformational changes in the protein. A previous proteolysis study using V8 protease
suggested an ATP- and ADP-dependent conformational change which protected Lon
from V8 digestion (73). The function of this proposed conformational change was not
known. By performing a similar study using tryptic digestion and correlating the results
with the steady-state kinetic data, we attempted to investigate the functional role of a
conformational change associated with nucleotide binding (55). Figure 2.6 shows the
tryptic digestion pattern of Lon alone or in the presence of various nucleotides over thirty
minutes. The presence of S2 peptide did not affect the digestion pattern indicating that
peptide does not induce any conformational change in Lon that is detectable by tryptic
digestion and that the presence of peptide does not affect the nucleotide dependent
conformational change (gels not shown). As shown in Figure 2.6a Lon’s digestion
pattern in the presence of ATP, GTP, CTP and UTP varies. Because Lon can hydrolyze
the nucleotides under the digestion conditions the data more likely represent Lon-NDP
resistance to tryptic digestion. The two prominent fragments in Figure 2.6a have
apparent molecular masses of 67 kDa and 26 kDa. The other fragments (45 kDa, 35 kDa,
23 kDa, and 7 kDa) only appear in the non-adenine containing nucleotides. This suggests
37
a
b
Figure 2.6 – Limited Tryptic Digestion of Lon in the Presence of Nucleotides. SDSPAGE visualized with Coomassie brillilant blue show 1.4 µM Lon monomer digested
with a limiting amount of trypsin and quenched with SBTI at the indicated times as
described in Materials and Methods. Lane 1 in both A and B show the molecular
markers in kilodaltons (from top to bottom): 183, 114, 81, 64, 50, 37, 26, 20.
38
that there is something unique about the adenine in terms of its affect on Lon’s structure.
We propose that Lon adopts a more compact conformation in the presence of adenine
nucleotides thus making it less susceptible to tryptic digestion. In order to verify the
unique affect of ATP two other adenine containing nucleotides, ADP and AMPPNP,
were incubated with Lon in tryptic digestion reactions. AMPPNP is a non-hydrolyzable
ATP analog that supports S3 peptide cleavage albeit at a slower rate than ATP. It should
mimic the effect of ATP binding as it is not hydrolyzed by Lon. Figure 2.6b shows that
the digestion patterns for the three adenine containing nucleotides are comparable and
that they substantially stabilize Lon to tryptic digest as compared to Lon with no
nucleotide. Lengthening the time of digestion did not significantly alter the pattern for
the adenine containing nucleotides. The same reactions shown in Figure 2.6 were
separated on a gradient gel, transferred to PVDF membrane, and sequenced by Edman
degradation (55). The first five amino acids of each fragment were identified and
matched against the Lon sequence. Table 2.3 summarizes the identified sequences and
trypsin cleavage sites.
Identifying Lon Fragments From Limited Tryptic Digestion Reactions. The relative
positions of the fragments compared to intact Lon were deduced from the sequencing
data shown in Table 2.3 and are summarized in Figure 2.7. The four domains contained
in each Lon monomer from the N- to C-terminus are (Figure 1.2) the N-terminal,
ATPase, substrate sensory and discriminatory (SSD), and the protease (23, 24, 31, 32).
Lon belongs to the AAA+ superfamily of ATPases. This family is based on multiple
sequence alignments which define a common ATPase module and encompasses a
broader range of proteins than the traditional AAA proteins (ATPases associated with
39
Table 2.3: Identification of the Tryptic Digestion Sites in Lon
Observed
molecular
mass (kDa)
67
45
35
26
23
7
Sequence
identifieda
Cleavage
site
Domains
included
Condition
AIQKE and
ELGEM
ELGEM
LSGYT
MNPER and
SERIE
ADNEN
LSGYT
A237/E241K783
A237-R587
L490-K783
M1/S6K236/K240
A588-K783
L490-R587
ATPase, SSD,
peptidase
ATPase, SSD
SSD, peptidase
Amino terminus
B
Peptidase
SSD
C
C
C
C
B
a
The sequence for the first five-amino acids of each Lon fragment was identified by
Edman degradation.
b
Detected in the absence or presence of NTPs.
c
Detected in the absence of adenine-containing nucleotides.
Adapted from Patterson et al. 2004
Figure 2.7 – Fragmentation of Lon resulting from limited tryptic digestion. The sizes of
the Lon fragments were estimated on the basis of their position on the SDS gel compared
sto the molecular mass markers. The relative positions of the fragments compared to the
intact Lon monomer were deduced from the sequencing data given in Table 2.3.
Adapted from Patterson et al, 2004 (55).
40
diverse cellular activities) which are a subfamily of the Walker type NTPases (25, 26).
Walker A and B are two consensus sequence motifs associated with nucleotide binding
(28, 29). Figure 2.7 shows the first cut by trypsin which separates the 26 kDa N-terminal
region from the rest of the domains. The sequencing summarized in Table 2.3 shows that
the N-terminus is resistant to any further digestion, which confirms the observation that
the N-terminus is also resistant to additional V8 protease digestion (73). The 67 kDa
fragment which contains the ATPase (Walker A and B), SSD, and protease domains is
further degraded by trypsin in the presence of the non-adenine containing nucleotides
(Table 2.3, Figure 2.7). The 35 kDa fragment includes both the SSD and protease
domain, the 23 kDa fragment includes only the protease domain, and the 7 kDa fragment
includes only the SSD domain. The 45 kDa fragment included both the ATPase and SSD
domain. No fragment including only the ATPase domain was detected. As shown in
Figure 2.6 gel B, when no nucleotide is bound to Lon the 45 kDa fragment (ATPase
domain) of Lon is rapidly degraded and is not detectable. This furthers supports the
hypothesis that the binding of nucleotide to Lon induces a closed conformation which
protects the enzyme from cleavage by trypsin.
Modeling the AAA+ Binding Site in Lon Using HslU as a Model. Because no complete
crystal structure exists for Lon that includes the nucleotide binding domain, we utilized
the crystal structure of HslU bound to dADP (PDB entry 1HT2) to model the global
effect of nucleotide binding as well as important residue interactions. Both Lon and
HslU share the AAA+ chaperone motif containing the Walker A and B nucleotide binding
consensus sequences as well as a SSD domain (25, 26). Examination of the structure of
the HslU nucleotide binding site (55) suggests that the N6 amino group on ADP functions
41
as a hydrogen bond donor that interacts with the carbonyl oxygen of both Ile 18 and
Val61 along the amide backbone. By aligning the sequence of Lon with this portion of
HslU, the Val61 residue was found to be conserved in Lon (Val 360). Furthermore,
Gln317 in Lon could serve the same function as Ile18 in HslU. These considerations
suggest that Lon binds to adenine nucleotides through two hydrogen bonding interactions
with the N6 amino group (55). The data in Figure 2.6a suggests that, CTP noticeably
stabilizes the 67 kDa fragment better than GTP and UTP. Both adenine containing
nucleotides as well as CTP stabilize the 67 kDa, ∆N-terminal-Lon fragment. Constrained
energy minimization calculations showed that the two amino groups on cytidine and
adenine are only 0.7 Å apart when overlaid (74). In other words, the amino groups on the
two nucleobases are in spatial proximity to one another (55, 74). The ability of CTP to
stabilize the 67 kDa fragment in the tryptic digest study is easily explained if it interacts
with Lon in a similar manner to ATP. The two nucleotides most likely make similar
contacts with Lon through hydrogen bond donors at the C6 positions (Figure 2.3) and
induce a closed conformation that is more resistant to tryptic digestion. If the closed
conformation were needed for optimal NTPase activity one would expect that ATP would
have the highest steady-state kcat,NTP value followed by CTP, and that GTP and UTP
would have comparable values. The steady-state NTPase data summarized in Tables 2.1
and 2.2 negate this hypothesis as UTP is hydrolyzed comparably to ATP and faster than
CTP and GTP. However, when the studies on the peptidase activity of Lon are taken into
account, the observations made in the ATPase activity studies correlate nicely.
Correlating the Peptide and Nucleotide Hydrolysis Activities of Lon. Lon hydrolyzes the
peptide substrate S3 (mixture of non-fluorescent S2, and fluorescent S1) only in the
42
presence of nucleotide substrate (40). Although the structural data alone is not
conclusive, given the similarities between Lon and HslUV as well as other AAA+
proteases, it is conceivable that Lon also couples ATP hydrolysis with proteolysis
through a translocation step. In order to provide more conclusive evidence for the
existence of an ATP-dependent peptide translocation step we characterized the NTPdependent peptidase activity of Lon using CTP, GTP, and UTP as activators. These
results were compared with the data reported previously for ATP and AMPPNP (40).
Under our assay conditions (0.5 mM NTP) the primary enzyme species present was Lon
bound to nucleotide. Therefore, the functional relationship between the kcat for NTP
hydrolysis and peptidase activation could be quantitatively measured. All nucleotides
tested supported peptide degradation to some extent (55). ATP was found to be the best
activator of peptidase activity followed by CTP > GTP ~ UTP > AMPPNP. ADP inhibits
peptidase activity in Lon presumably by acting as a competitive product inhibitor of the
ATPase activity (40). In the case of the hydrolysable nucleotides, this trend of the ability
of the nucleotides to support peptide degradation is identical to the nucleotide’s ability to
induce the closed conformation in Lon which protects it from tryptic digestion by
stabilizing the 67 kDa fragment (Figure 2.6a). It is possible that the conformational
change induced by the binding of nucleotide is necessary for peptide translocation to the
protease active site. Therefore, the nucleotides which stabilize that conformation (ATP
and CTP) would enhance the ability of Lon to degrade peptide. AMPPNP and ADP are
not hydrolyzed by Lon but induce the same conformational change as ATP (Figure 2.6b).
Because neither AMPPNP nor ADP support maximal peptide hydrolysis both binding
and hydrolysis of nucleotide must be necessary for peptide translocation.
43
Proposed Model for the Role of ATP Binding and Hydrolysis in the Peptidase Activity of
Lon. By incorporating the information gleaned from the steady-state characterization of
the NTPase and peptidase activities of Lon in the presence of various nucleotides, the
tryptic digest study, and previous kinetic characterization performed in the lab (40, 53,
55) we formulated a model for the role of ATP binding and hydrolysis in the peptidase
activity of Lon. The structural changes noted in HslU as a result of nucleotide binding
were incorporated as HslU is a reasonable model for the ATPase motif in Lon due to the
high sequence homology (29, 61). Figure 2.8 depicts our proposal for how the ATPdependent conformational change, followed by hydrolysis facilitate the movement of
peptide to the active site in Lon (55). Although Lon is thought to be hexameric, it is
represented in Figure 2.8 as a dimer for clarity. The protease domain of Lon crystallized
with the monomeric subunits organized in a barrel shape with a central cavity containing
the active site serine (32). This is similar to the organization of other AAA+ ATPdependent proteases (26). The three domains of Lon (protease, SSD, and ATPase) are
connected by black linkers and shown in Figure 2.8 in white, green, and blue
respectively. Enzyme form I shows an open conformation of free Lon with no nucleotide
bound. Upon nucleotide binding the enzyme undergoes a conformational change that
makes Lon more resistant to tryptic digestion (form II, Figure 2.8). We propose this
conformational change rearranges Lon in such a way as to promote peptide delivery.
However, the peptide cannot gain full access to the active site without hydrolysis of the
nucleotide (form III, Figure 2.8). This is supported by the fact that ADP and AMPPNP
which are not hydrolyzed by Lon, can still induce a similar conformational change as
ATP. Once the peptide is hydrolyzed (form IV, Figure 2.8) and ADP is release, Lon
44
Figure 2.8 – Model proposed for the different enzyme forms in Lon that couple ATP
binding and hydrolysis in activating peptide hydrolysis. This model is proposed on the
basis of the structural similarities shared by Lon and HslU. An ATPase-dependent
peptide translocation is proposed in this model. The α/β-subdomain and the α-helical
subdomain of the AAA+ motif are in blue and green, respectively. The protease domain
is in white. The protein domains and subdomains are connected by flexible linkers.
Adapted from Patterson et al. 2004.
45
relaxes back to the open conformation to repeat the cycle. This model predicts that ATP
hydrolysis occurs prior to peptide cleavage, and that nucleotides that cannot induce the
closed conformation should be poor activators of peptide cleavage. Based on the steadystate kcat values associated with peptide cleavage, the hydrolysable nucleotides that have
hydrogen bond donating properties at the C6 position (Figure 2.3) are the best activators
of Lon’s peptidase activity (55). The timing of ATP and peptide hydrolysis can be
elucidated using pre-steady-state kinetic methods. If ATP hydrolysis occurs prior to
peptide cleavage the pre-steady-state rate constant should be higher than that associated
with peptide cleavage. The determination of the timing of events will clarify the
mechanistic details of Lon’s activities.
46
CHAPTER 3
ASSESING THE TIMING OF MECHANISTIC EVENTS IN ESCHERICHIA COLI
LON PROTEASE THROUGH CHARACTERIZATION OF ITS PRE-STEADY
STATE ATPase ACTIVITY
47
Introduction
Lon protease couples the energy generated from ATP hydrolysis with the
degradation of protein or peptide substrates (1). The steady-state characterization of E.
coli Lon protease’s ATPase activity was described in the previous chapter. The turnover
numbers (kcat) associated with peptide stimulated and intrinsic ATPase activity are a
compilation of all steps in the reaction related to ATP hydrolysis including binding,
conformational changes, hydrolysis, and product release. The turnover numbers are
limited by, and therefore reflective of, the slowest step in the pathway. Although steadystate kinetic characterization provides some information about the mechanism as a whole,
individual rate constants cannot be measured. In order to monitor individual steps along
the ATPase pathway, we instead utilized pre-steady-state kinetic techniques. Pre-steadystate kinetic analysis of an enzymatic system allows for the examination of activity on a
time scale of a single enzyme turnover.
Two methods typically used to mix the enzyme and substrate are stopped flow
and rapid quench (75). Although both instruments can mix the enzyme and substrate in
approximately one millisecond, stopped flow experiments require an optical signal to
monitor changes in fluorescence, absorbance, or light scattering. On the other hand,
rapid quench experiments do not rely on an optical signal. Instead, radiolabeled
substrates are generally utilized to monitor an enzymatic reaction in a rapid quench
experiment. In order to stop the reaction in this type of experiment, a chemical
denaturant such as acid is mixed with the reaction after a designated period of time. Each
individual time point is collected and the amount of product generated measured by either
chromatographic or electrophoretic separation. Although the rapid quench experiments
48
are discontinuous and thus more labor intensive than stopped flow experiments, they
provide direct information about the rate of the chemical reaction as well as information
about the absolute concentrations of the reactants and products (76). Three types of time
courses are typically detected in the first turnover in a pre-steady-state experiment where
product formation is being monitored (75). These are a burst phase, lag phase, or linear
phase and the time courses can be fit using an equation containing an exponential
function followed by a linear function from which the rate constants can be extrapolated
with the exception of the linear phase. Each phase can indicate some information
regarding the rate-limiting step of the reaction. Because a burst phase is a result of rapid
product formation followed by a slower turnover it indicates that the rate limiting step is
after the hydrolysis. The lag phase is a result of the buildup of a reaction intermediate
prior to turnover which indicates that the rate limiting step precedes hydrolysis. Finally
the linear phase indicates that hydrolysis or the step prior to hydrolysis is rate limiting for
turnover of the enzyme.
This chapter describes the various rapid quench experiments designed to probe presteady-state ATP hydrolysis in Lon protease. The experiments were performed under
pseudo-first-order conditions which means that the concentration of the substrate [α32
P]ATP was in excess of the enzyme concentration. Because excess substrate is present
in the reaction both the first round of ATP hydrolysis as well as steady-state ATP
hydrolysis is detected in the time courses. Scheme 3.1 shows a schematic of the rapid
quench. The Lon solution is loaded into sample loop B and the [α-32P]ATP solution
loaded into sample loop A. The two are mixed in the reaction loop for a designated
49
Scheme 3.1 – KinTek Corporation Rapid Quench Schematic. The Lon solution is loaded
into sample B and the [α-32P]ATP loaded into sample B. There are seven reaction loops
of varying length to allow for different time points. The acid quench denatures the
enzyme at the specified times through the quench line, and each individual time point is
collected through the exit line.
50
period of time (0-3 s) and quenched with acid through the quench line. The individual
time points are then collected from the exit line, and [α-32P]ATP and [α-32P]ADP are
separated using thin layer chromatography. The radioactive spots on the TLC plates are
then visualized using phosphorimaging.
We found that the pre-steady-state ATPase activity in Lon protease displays burst
kinetics. This indicates that a step following chemistry, such as product release or a
conformational change, is rate limiting for the turnover of the enzyme. However, the presteady-state burst activity was found to be unusual in that the amplitude was only half
that of the concentration of Lon in the reaction. A filter binding assay was performed to
ensure that all enzyme monomers in the reaction were capable of binding nucleotide.
Furthermore, a pulse-chase experiment confirmed that every Lon monomer could
hydrolyze ATP. This indicated that the high- and low-affinity ATPase sights hydrolyze
ATP at drastically different rates. When the pre-steady-state data from the ATPase
activity of Lon was compared with the pre-steady-state peptidase activity it was clear that
ATP was being hydrolyzed prior to peptide cleavage (77). The majority of the
experiments described in this chapter were published in reference (77), and the
correlation of the peptidase and ATPase activity is described in more detail.
51
Materials and Methods
Materials. ATP was purchased from Sigma whereas [α-32P] ATP was purchased from
Perkin Elmer or ICN Biomedical. Tris, HEPES and PEI cellulose TLC plates were
purchased from Fisher.
General Methods. Protein purification procedures were performed as described
previously (78). All enzyme concentrations were reported as Lon monomer
concentrations. All reagents are reported as final concentrations, and experiments were
performed at 37 °C unless otherwise indicated.
Chemical Quench ATPase Activity Assays. The pre-steady-state time courses for ATP
hydrolysis were measured using a rapid chemical quench-flow instrument from KinTek
Corporation. All solutions were made in 50 mM HEPES buffer pH 8.1, 5 mM DTT, 5
mM Mg(OAc)2, 75 mM KOAc. A 15 µL buffered solution of Lon monomer (2–9 µM),
with and without 500 µM S2 or 10 µM casein, was rapidly mixed with a 15 µL buffered
solution of ATP (≤ 200 µM) containing 0.01% of [α-32P]ATP at 37 °C for varying times
(0-3 s) before quenching with:
- 0.5 N formic acid and then extracted with 200 µL of phenol/chloroform/isoamyl
alcohol pH 6.7 (25:24:1).
- 1 N HCl and then neutralize with 4 M Tris, 2 N NaOH. Because the loop volumes vary,
the amount of the neutralization solution varies, so the specific amounts were determined
and listed in the chart below.
52
Loop
Recovered Volume
(µL)
Acid Volume
(µL)
1
2
3
4
5
6
7
102
115.5
137.5
150.8
169.3
184.8
216
72
85.5
107.5
120.8
139.3
154.8
186
Neutralization
solution volume
(µL)
21
25.7
32.3
36.2
41.8
46.4
55.8
The neutralized reactions were spotted on pH paper to ensure that they were at pH 8. The
reactions were then extracted with 200 µL of phenol/chloroform/isoamyl alcohol pH 6.7
(25:24:1).
-250 mM EDTA, 100 mM Tris pH 10, then add 10% SDS for a final concentration of 1%
SDS (refer to chart above for recovered volume).
For all quenches, a 3 µL aliquot of the aqueous solution was spotted directly onto a PEIcellulose TLC plate (10cm x 20cm), and the plate was developed in 0.75 M potassium
phosphate buffer (pH 3.4) for the acid quenches and 0.5 M LiCl, 1 N Formic acid for the
EDTA/SDS quench to separate ADP from ATP. The relative amount of radiolabeled
ADP and ATP at each time point was quantified by a Cyclone Phosphor imager (PerkinElmer Life Science). To compensate for the slight variations in spotting volume, the
concentration of the ADP product obtained at each time point was corrected for using an
internal reference as shown in eq 1.

ADPdlu
[ ADP] = 
 ATPdlu + ADPdlu

 × [ ATP ]

(1)
53
All assays were performed at least in triplicate and the average of those traces used for
data analysis. The burst amplitudes and burst rates were determined by fitting the kobs
data from 0 to 400 ms with eq 2.
Y = A * exp − k burst t + C
(2)
where t is time in seconds, Y is [ADP] in µM, A is the burst amplitude in µM, kburst is the
burst rate constant in s-1, and C is the end point. The observed steady-state rate constants
(kss,ATP) were determined by fitting the data from 600 ms to 1.8 s with the linear function,
Y= mX +C, where X is time, Y is [ADP] / [E], m is the observed steady-state rate
constant in s-1, and C is the y-intercept. Data fitting was accomplished using the
nonlinear regression program KaleidaGraph (Synergy).
Filter Binding Assay. Two to five µL of a 35 µM stock of Lon was incubated with 10
µM of [α32P] ATP in 30 µL of 50 mM HEPES pH 8.1, 5 mM Mg(OAc)2, 75 mM KOAc,
2 mM DTT at 37 °C for 20 minutes to convert all ATP to ADP. The reactions were then
chilled on ice and 3 µL of the reactions (performed in triplicate) were spotted onto a
piece of nitrocellulose mounted onto a dot-blot apparatus (BioRad) as described by
Gilbert and Mackey (63, 79). Each spot was washed with 10 µL of cold buffer and dried
under vacuum for 30 minutes. In the absence of vacuum, the nitrocellulose was spotted
with 2 µL of each reaction and then air-dried. The radioactive counts at each spot were
quantified by PhosphorImaging.
Bradford Assay. Two, four, six, and eight µL of 1 mg/mL BSA was diluted to 100 µL
with 0.1 M NaCl in disposable 2 mL plastic cuvettes. Additionally two, four, and six mL
of the stock of Lon was also diluted to 100 mL in the same manner. 1 mL of Bradford
dye was added to the cuvettes, mixed well, and allowed to incubate for 5 min.
54
Absorbance readings at 595 nm were recorded for all the samples. The A595 for the BSA
samples was plotted versus the volume of protein to generate a standard linear curve.
The linear function was used to solve for the corresponding mg/mL of Lon which was
converted to units of concentration by using the monomeric molecular weight, 89 kDa.
Pulse Chase ATPase Activity Assays. The
pre-steady-state
time
courses
of
ATP
hydrolysis were also measured using a pulse chase experiment on the rapid quench. Lon
(+/- 0.5 mM S2) was rapidly mixed with radiolabeled ATP at 37 oC for 0 to 1.8 seconds,
followed by a 10 mM unlabeled ATP chase for 60 seconds before quenching with 0.5 N
formic acid.
The amount of ADP produced at each time point was quantified as
described in the chemical quench assay (see above). The burst amplitude (A) and burst
rate constant (kburst) were determined from the time courses by fitting the data from 0 to
400 ms with eq. 2.
55
Results and Discussion
Control Reactions Demonstrate Rapid Quench Functions Properly. The goal of the
studies described in this chapter was to monitor the first round of ATP hydrolysis by Lon
protease. In order to accomplish this, pre-steady-state kinetic experiments were used
because the ATPase reaction could be monitored on a millisecond time scale. The presteady-state ATPase activity of Lon was monitored using a rapid quench (KinTek
Corporation) because the radiolabeled substrate, [α-32P]ATP, used in the steady-state
experiments described in Chapter 2 could be used to visualize the reaction. The rapid
quench instrument uses a computer-controlled servo motor drive to mix reaction
components on a millisecond time scale so that the first turnover of ATP cleavage can be
visualized. Before Lon reactions were monitored using the rapid quench, I performed
control reactions to insure the instrument was working properly. First, the various loop
volumes in the instrument were calibrated according to the manufacturer’s instructions
using a [γ-32P]ATP solution. Briefly a stock solution of the labeled nucleotide was made
with a cpm count between 10,000 and 20,000 cpm. The various loops were filled with
the stock solution and collected. The radioactive counts (cpm) can then be directly
correlated to the volume of the loops. My calibration results correlated well with the
initial calibration done when the instrument was purchased. A manufacturer suggested
control reaction was then performed which monitored the base catalyzed hydrolysis of
benzylidenemalononitrile (BMN) (80). This reaction, shown in Scheme 3.2, is known to
occur with a second order rate constant of 140 M-1s-1. The kinetics of hydrolysis were
measured by monitoring the decrease in absorbance at 310 nm. When the kinetic data are
plotted, the change in absorbance with time should fit a single exponential decay with a
56
1 M NaOH
CH
CN
+
C
H
CH2(CN)2
O
C
NC
Scheme 3.2 – The base catalyzed hydrolysis of benzylidenemalononitrile (BMN) can be
monitored by the decrease in absorbance at 310 nm.
57
rate constant approaching 70 s-1. Figure 3.1 shows the control reaction for the rapid
quench performed at room temperature. When the data in Figure 3.1 were fit with
equation 1, the rate constant obtained was 59 + 0.03 s-1 which approaches the known
value (70 s-1). As both the calibration of the rapid quench and control reaction resulted
with reasonable data, the Lon ATPase reaction was investigated.
Optimizing Rapid Quench Reaction Conditions. Because the pre-steady state activity of
Lon had never been monitored, all of the experimental conditions including the
concentrations of enzyme and substrate, the type of denaturing quench, the distribution of
the time points, and the TLC developing buffer conditions used to separate ATP and
ADP needed to be optimized. I first examined the first turnover of ATP hydrolysis by
Lon under pseudo-first order conditions. Under these conditions, the concentration of
ATP is in excess over the concentration of Lon, which ensured that the same enzyme
form (Lon:ATP) and intermediates as seen in the steady-state were being monitored.
Furthermore, as the pre-steady-state reaction time courses were monitored into steadystate turnover, the steady-state rate constant obtained from the rapid quench experiments
could be compared to the known values. This served as a control that ensured that Lon
was functioning normally when the new experimental technique was used. The
radioactive assay used to monitor the steady-state ATPase activity of Lon utilized formic
acid as a quench for the reactions. The reactions were then spotted on PEI-cellulose TLC
plates and [α32-P]ATP and [α32-P]ADP were separated by developing the plate in 0.3 M
KPi pH 3.4. In the pre-steady state reaction time courses, much less [α32-P]ADP is being
formed at the various time points. In order to facilitate the separation of the di- and
58
0.7
absorbance (310 nm)
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0
0.02
0.04
0.06
0.08
0.1
time (s)
Figure 3.1 – Base catalyzed hydrolysis of benzylidenemalanitrile at room temperature as
monitored using the Rapid Quench (KinTek Corp). The data were fit using a single
exponential decay equation which resulted in a rate constant of 59 + 0.03 s-1.
59
triphosphate, the concentration of the developing buffer was increased to 0.75 M KPi pH
3.4 for the acid quenched reactions. As suggested in the literature, the developing buffer
used for an EDTA quenched reaction was 0.5 M LiCl, 1 N formic acid (81, 82).
In some enzyme systems, identical reactions quenched under differing conditions
(i.e. HCl vs. formic acid) displayed differing time courses (83). To ensure that this
discrepancy was not noted in the Lon ATPase reaction a variety of denaturing quench
solutions were tested including 0.5 N formic acid, 1 N HCl, and 250 mM EDTA in 100
mM Tris pH 10 to ensure they resulted in identical time courses. By using a denaturing
quench both Lon:ADP as well as ADP are detected as indicated in Scheme 3.3. Because
the reaction was being monitored on a millisecond time scale, I designed an experiment
that determined whether these quenches were inactivating Lon within 5 ms. To this end
15 µL of buffer was rapidly mixed with 15 µL of Lon or control buffer for 5 ms before
quenching. The quenched reaction was expelled into a eppendorf tube containing 200
µM [α32-P]ATP. The reactions were then developed using strong anion exchange thin
layer chromatography to separate [α32-P]ATP from [α32-P]ADP and visualized using
PhosphorImaging. The quench was considered to be adequately inactivating Lon if the
buffer control and quenched Lon reaction had the same amount of [α32-P]ADP formation.
Figure 3.2 depicts the difference between the control and quenched Lon reaction for the
various denaturing quenches tested. This experiment showed that the formic acid quench
resulted in the least amount [α32-P]ADP formation as well as the smallest difference
between the reaction with and without enzyme. The HCl quench adequately inactivated
the enzyme, however overall more [α32-P]ADP was produced. The basic (pH 10)
EDTA/SDS quench did not stop the reaction as well as the two acidic quenches. Because
60
Lon + ATP
Lon:ATP
Lon:ADP
Lon + ADP
Scheme 3.3 – Minimal kinetic mechanism for ATP hydrolysis by Lon protease. The box
encloses both released ADP as well as Lon bound to ADP because the denaturing quench
allows for the detection of both intermediates.
5
EDTA / SDS
[ADP]
control
- [ADP]
Lon
(µM)
4
3
2
HCl
1
Formic Acid
0
Figure 3.2 – Effectiveness of the various denaturing quenches in inactivating Lon
protease within 5 ms. The bars indicate the difference between the quenched Lon
reaction as compared to a buffer only control. The quenches with higher bars do not
inactivate Lon as well as the others.
61
formic acid is not as strong of an acid as HCl, it does not need to be neutralized. This
shortens the procedure and therefore we selected formic acid to be the standard acid
quench for use in the Lon system. It is known that Lon binds tightly to ADP (Ki = 300
nM) and that ADP is a potent inhibitor of the enzyme (40). Therefore, to ensure that all
of the [α32-P]ADP from the Lon:ADP species was being detected a phenol-chloroform
extraction was added post-acid quenching to separate the enzyme from the nucleotide
species. The phenol chloroform extraction ensures that ADP is not still tightly bound to
Lon. The formic acid quench was used to determine optimal concentrations of Lon and
ATP for use in monitoring reactions with saturating amounts of ATP present. Because so
little [α-32P]ADP is being produced on the millisecond time scale compared to the
amount of [α-32P]ATP present, the resolution of the pre-steady-state ATPase time
courses was very difficult. After many attempts under various conditions, I found that at
least 5 µM Lon and only concentrations of [α-32P]ATP up to 200 µM were needed for the
reactions to be resolved at 37 °C. At any higher concentrations of ATP, the small amount
of ADP product could not be resolved from the relatively large amount of ATP. Even
under the optimized conditions, multiple trials were always performed and the data
averaged. The error in the time courses was in part due to the inherent error generated
from using a discontinuous assay as well as resolution issues. Lowering the temperature
to 25 °C slowed the reaction slightly, but did not produce significantly cleaner data to
warrant using the non-physiological temperature. Because the intrinsic ATPase activity
of Lon is slower than the peptide or protein stimulated ATPase activity, resolution of
reactions using less that 200 µM [α-32P]ATP was impossible. Instead, I fully
characterized the pre-steady-state S2 peptide stimulated ATPase activity so that the
62
results could be combined with that gleaned from the pre-steady-state peptidase activity
of Lon to generate a detailed kinetic mechanism for the enzyme.
Using the optimized reaction conditions where Lon was saturated with ATP,
control reactions were performed to ensure that the different types of quenches resulted in
identical time courses. Figure 3.3 shows a comparison of a S2 peptide (Figure 1.6)
stimulated Lon ATPase time course at 37 ºC using the three denaturing quenches. The
formic acid and HCl quenched reactions were processed identically with the exception of
the HCl quenched reactions being neutralized. The EDTA/SDS quenched reaction had
no phenol chloroform extraction performed and was developed in a different running
buffer. These slight differences could account for the slightly lower production of [α32
P]ADP in the later time points of the EDTA/SDS quenched reaction. In each case Lon
displays a pre-steady-state burst of [α-32P]ADP production. This type of activity
indicates that a step following the hydrolysis of ATP is rate limiting for turnover of the
enzyme. Previous steady-state studies on Lon had suggested that ADP release was the
rate limiting step which would be consistent with the observed pre-steady-state burst (3840). The pre-steady-state burst amplitude is a function of the rate of ATP hydrolysis
relative to the rate of product release, the amount of active enzyme, and the internal
equilibrium constant for ATP hydrolysis at the active site (76, 84). As long as one
hundred percent of the enzyme is active, the equilibrium favors product formation, and
the rate of product release is slower than hydrolysis, the burst amplitude should equal the
amount of enzyme present in the reaction (76). A burst followed by steady state turnover
like those shown in Figure 3.3 are typically fit with a burst equation which contains a
single exponential function followed by a linear function (equation 3).
63
2.5
32
[α- P] ADP / [Lon] ( µM)
2
1.5
1
0.5
0
0
0.5
1
1.5
2
2.5
3
3.5
Time (s)
Figure 3.3 – Pre-steady-state S2 stimulated ATPase activity of Lon protease quenched
with 0.5 N formic acid (●), 1 N HCl (■), and 250 mM EDTA in 100 mM Tris pH 10 / 1%
SDS (♦).
64
[ ADP ] /[ E0 ] = A * (1 − e − k burst *t ) + k ss , ATP * t
(3)
where [E0] is the concentration of Lon monomer, A is the burst amplitude, kburst is the
burst rate constant, t is time, and kss,ATP is the steady state rate constant for ATP
hydrolysis. This equation relies on the rapid accumulation of the Lon:ATP complex
meaning that substrate binding should not limit the reaction under the experimental
conditions used. As discussed in Chapter 6, I have measured the rate of ATP binding to
Lon and found that it approaches diffusion control. The burst amplitude shown in Figure
3.3 is unusual because in all three cases it only approaches half of the enzyme
concentration. In addition there is a transition period between 0.5 – 1 s before the steadystate activity is reached. For simplicity Figure 3.4 shows only the formic acid quenched
S2 stimulated ATPase activity and outlines the unusual regions in the time course. The
black line shows the fit of data with the standard burst equation. This equation obviously
does not converge well most likely due to the transition phase between the burst and
steady-state activities. Because the pre-steady-state burst activity of Lon is not standard
and thus not described by the burst equation (3), the data was instead split into a single
exponential pre-steady-state phase up to 0.6 s, and a linear steady-state phase for fitting
purposes. Figure 3.5a shows the pre-steady-state ATPase time courses for Lon with no
peptide substrate (intrinsic), 500 µM S2 peptide substrate, and 20 µM casein at 100 µM
ATP. The data was fit with a single exponential equation up to 0.6 s shown in Figure
3.5b and a linear equation from 1 s to 3 s. The kinetic parameters extrapolated from the
data fitting including the burst amplitude, burst rate
65
4
1
3.5
[ADP] / active site
( µM)
0.8
0.6
[ADP] / active site
( µM)
3
0.4
0.2
2.5
0
2
0
0.2
0.4
0.6
time (s)
0.8
1
Steady-state
1.5
1 burst
0.5
transition
0
0
0.5
1
1.5
2
2.5
3
3.5
time (s)
Figure 3.4 – Pre-steady-state S2 stimulated ATPase activity of E. coli Lon Protease. The
black line shows the data fitting with the burst equation (3). The burst (0 – 0.5 s),
transition (0.5 – 1 s), and steady state phases (1 – 3 s) of the time course are indicated.
The inset zooms in to show the burst and transition phases more clearly.
66
a
[ADP]/active site, µM
2.5
2
1.5
1
0.5
0
0
0.5
1
1.5
2
2.5
3
time (s)
b
2
0.3
1.5
0.2
1
0.1
0.5
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
[ADP] , µM
[ADP] / active site, µM
0.4
0
time (s)
Figure 3.5 – Pre-steady-state time courses of ATP hydrolysis by Lon (a &b) in the
absence of peptide or protein substrate (●), in the presence of saturating (500 µM)
amounts of S2 peptide (■), and in the presence of saturating (20 µM) amounts of casein
(♦). The data from 600 ms to 3 s was fit with a linear equation to get the steady-state rate
constant (kss, ATP). Figure 3.5b zooms in on the burst region of the time courses up to 400
ms which was fit with a single exponential equation to obtain values for the burst rate and
amplitude. The kinetic parameters are summarized in Table 3.1.
67
constant, and the steady-state rate constant, are summarized in Table 3.1. As one would
expect from previous studies (1, 40), the steady-state ATPase activity in the absence of
peptide (intrinsic) is approximately two fold less than the S2 peptide and casein
stimulated activity. The pre-steady-state burst amplitude and burst rate for all three
conditions are comparable (Table 3.1). This indicates that the presence of peptide or
protein substrate does not affect Lon’s pre-steady-state ATP hydrolysis activity. The
burst rate constants are also comparable in the presence and absence of peptide or protein
substrate and faster than the steady-state turnover number of the enzyme as one would
expect. However, the stoichiometry of the burst amplitude is only half that of the enzyme
present in the reaction. The ATPase reaction in the presence of S2 peptide could be
correlated to the peptidase activity of the enzyme so it was examined in more detail. The
Km for ATP determined using steady-state methods is approximately 50 µM. The rapid
quench experiments shown in Figure 3.5 contained 100 µM ATP because the signal to
noise ratio increases with increasing ATP concentrations. In this case “noise” is a result
of using a saturating concentration (> 100 µM) of ATP in the reaction while at best only
2.5 µM ADP is formed. Because the sub-stoichiometric burst amplitude measured in the
acid quench experiments could be attributed to the relatively low amount of nucleotide
present, the concentration of ATP was varied. The amplitude is expected to increase with
higher nucleotide concentrations if this hypothesis were correct. Figure 3.6 shows presteady-state ATP hydrolysis time courses at increasing concentrations of ATP. The time
courses were fit with a single exponential equation from 0 – 400 ms to obtain the burst
amplitude and rate constant and a linear equation from 600 ms to 3 s to obtain the steady-
68
Table 3.1: Summary of Kinetic Parameters Obtained From the Fitting of the Data in
Figure 3.5 a & b
kss,ATP
kburst
burst amplitude
intrinsic ATPase
0.23 s-1
7.6 + 1.3 s-1
1.92 + 0.12 µM
+20 µM casein
0.66 s-1
12.2 + 3.2 s-1
1.8 + 0.2 µM
69
+500 µM S2
0.69 s-1
6.5 + 1.4 s-1
2.06 + 0.15 µM
state rate constant. These parameters were then plotted versus the concentration of ATP
in Figures 3.7a, b, and c. Figure 3.7a shows that the steady-state rate constant kss,ATP has
a hyperbolic dependence on ATP concentration, which was then fit using the MichaelisMenten equation. The kinetic parameters are shown in Table 3.2. As one would expect,
the kcat,ATP and Km,ATP are comparable to the values obtained in the steady-state
characterization of Lon described in the previous chapter. Figure 3.7b shows that the
dependence of the burst amplitude on increasing concentrations of ATP is also
hyperbolic. The kinetic parameters are summarized in Table 3.2. The KATP (22 µM)
which is the concentration of ATP required to reach one half the maximal burst amplitude
is approaching the Kd of the low-affinity ATPase site (10 µM) (38). The maximal burst
amplitude is 2.2 µM, which is equal to approximately half of the 5 µM enzyme present in
the reaction. The burst rates are plotted versus the concentration of ATP in Figure 3.7c.
There appears to be no dependence of kburst on ATP concentration for. As both of these
phenomena were unusual, they were investigated further.
A Filter Binding Assay Demonstrates that all Lon Monomers Bind Nucleotide. A full
burst reflects the active enzyme concentration as long one hundred percent of the enzyme
is active, the equilibrium favors product formation, and product release is slow (76).
Therefore, an obvious explanation for the half burst seen in the pre-steady-state is that all
of the enzyme in solution is not capable of binding and or hydrolyzing ATP. I tested for
these possibilities using a filter binding assay and a pulse-chase rapid quench experiment.
Filter binding assay conditions for the Lon system were optimized using [α-32P]ATP.
This type of experiment can distinguish the amount of radiolabeled nucleotide bound to
Lon. A Bio-Rad Dot-Blot apparatus (Figure 3.8a) was purchased because the rubber
70
10
1.6
8
1.2
6
0.8
4
0.4
2
[ADP] (µM)
ADP / active site
(µM)
2
0
0
0.5
1
1.5
2
time (s)
Figure 3.6 – Pre-steady-state S2 stimulated ATPase activity at increasing concentrations
of ATP (+) 5 µM (U) 10 µM (◊) 25 µM (□) 50 µM (x) 100 µM (○) 200 µM.
3.7a
1.5
0.9
k
ss,ATP
-1
(s )
1.2
0.6
0.3
0
0
50
100
150
200
[ATP] (µM)
Figure 3.7 - (a) Data from 600 ms to 3 s in Figure 3.6 were fit with a linear function to
provide the steady-state rates of ATP hydrolysis at varying [ATP]. The kss,ATP values
were obtained by dividing the steady-state rates by [Lon]. Plotting the kss,ATP value
against its specific [ATP] yields a hyperbolic plot which was best fit with equation 1
from Chapter 2. The kinetic parameters obtained from the fit are summarized in Table
3.2.
71
b
burst amplitude ( µM)
2.5
2
1.5
1
0.5
0
0
50
100
150
200
250
300
200
250
300
[ATP] (µM)
c
20
k
burst
-1
(s )
15
10
5
0
50
100
150
[ATP] (µM)
Figure 3.7 – (b) Burst amplitudes were determined by fitting the data from 0 to 600 ms in
Figure 3.6 with equation 2. As summarized in Table 3.2, the maximum burst amplitude
obtained from the fit was 2.2 µM, which corresponds to ~44% of the enzyme present in
the reaction. (c) The burst rates of ATP hydrolysis were determined in the same manner
as in b, and the average kburst = 11.3 + 3.3 s-1 (Table 3.2). (a,b,c) Each time point was
performed at least 3 times and the averaged data were reported. The error bars represent
the error of the fit for each data point.
72
Table 3.2: Summary of the Pre-Steady-State Kinetic Parameters Associated with S2
Stimulated ATP Hydrolysis by Lon Protease
kcat,ATP
Km,ATP
KATP
kburst
Burst amplitude
2.0 + 0.23 s-1 a
143 + 30 s-1 a
22 + 16 µM b
6.5 + 1.4 d (11.3 + 3.3) c (18 + 2) e s-1
2.06 + 0.15 d (2.2 + 0.1) b (4.1 + 0.1) e s-1
a
These values were obtained by fitting the data in Figure 3.7a with the Michaelis Menten equation.
These values were obtained by fitting the data in Figure 3.7b with the Michaelis Menten equation.
c
These values were obtained by fitting the acid quenched time courses shown in Figure 3.7c with equation
3.
d
These values were obtained by fitting the data in Figure 3.5 with equation 2.
e
These values were obtained by fitting the pulse-chase data in Figure 3.10 with equation 2.
b
73
gasket created a tight seal which was necessary for reproducible results. This apparatus
basically resembles a 96 well plate on the top and a nitrocellulose membrane can be
sandwiched between the top piece and the rubber gasket. When all the components of the
apparatus are assembled it is hooked up to a vacuum so that the reaction solution can be
passed through the membrane and washed with the aid of the suction. Figure 3.8 shows a
cartoon depiction of the Dot-Blot components. The amount of reaction to be spotted, the
volume and number of rinses, as well as the buffer conditions were all optimized. The
presence of 0.01% Tween 20 detergent was found to reduce nonspecific binding. The
procedure described below uses the optimized conditions.
In order to test Lon’s ability to bind nucleotide, increasing amounts of the protein
stock solution (2-5 µL) were incubated with 10 µM [α-32P]ATP at 37 °C for 20 minutes
in order to convert all of the [α-32P]ATP to [α-32P]ADP. After chilling the reactions on
ice to slow the off rate of the nucleotide, they were spotted on the nitrocellulose
membrane mounted on the Dot-Blot apparatus. Because nitrocellulose membrane binds
proteins, only Lon:[α-32P]ADP should remain bound. The spots were washed with buffer
to eliminate any unbound nucleotide, and the radioactive counts on the membrane were
visualized with PhosphorImaging. The concentration of radiolabeled nucleotide at each
spot was calculated by using spots of known [α−32P]ATP concentration as a reference.
Figure 3.9 shows the picomoles of [α-32P]ADP plotted versus the volume of Lon in each
reaction.
Each Lon monomer has one nucleotide binding site, so the concentration of
Lon can be inferred from the slope of this plot. This concentration of Lon was then
compared to the concentration determined from the standard Bradford assay described in
the Materials and Methods. As the concentrations from the two assays were comparable,
74
a
b
E●ADP
100% ATP
Spot
3x
Lon (µL)
Figure 3.8 – (a) Bio-Rad Dot-Blot assembly. The sample template (1), gasket (2), and
gasket support plate (3) the Bio-Dot attaches to the vacuum manifold base (4) the
membrane (5) is placed on the gasket. (b) representative phosphorimaged nitrocellulose
membrane with increasing amounts of Lon bound to radiolabeled nucleotide next to
control spots.
75
200
32
α- P ADP bound
(pmole)
y = 5.486 + 36.139x R= 0.99732
150
100
50
0
0
1
2
3
4
5
6
µl Lon monomer
Figure 3.9 – Determining the concentration of active Lon monomer (35 µM as
determined by the Bradford assay) using a filter binding assay. The amount of Lon:[α32
P]ADP was determined as described in the Materials and Methods. Plotting the amount
of Lon:[α-32P]ADP versus the volume of the enzyme stock yielded a straight line that
was fit with the linear equation y = mx + c where m equals 36 pmol of ADP/µL of Lon
(36 µM Lon monomer).
76
all of the Lon monomer in solution must be capable of binding nucleotide.
A Pulse-Chase Experiment Confirms that All Lon Monomers Hydroyze ATP. Because the
reaction used in the filter binding assay was pre-incubated to allow for hydrolysis of
ATP, it was assumed that the enzyme not only bound ATP but hydrolyzed it as well.
However, the manner in which the data was quantified did not distinguish between the
radiolabeled nucleotide di- and tri-phosphate. Therefore, a pulse-chase experiment was
used to determine whether all the Lon monomers were capable of hydrolyzing ATP. As
shown in Scheme 3.4, this experiment differs from the acid quench experiments
described in the chapter. A second quench delay is added to the experiment to allow for
the labeled nucleotide bound in the reaction loop to be processed for a specified amount
of time before the reaction is quenched with acid. The excess unlabeled nucleotide is
introduced to prevent additional labeled nucleotide from binding in the duration of the
delay. As the length of this delay was increased from 10 s to 3 min, the burst amplitude
of the reaction increased. In fact, the burst amplitude was stoichiometric to the amount
of enzyme in the reaction only when the delay was at least 60 s. Figure 3.10a shows a
comparison of Lon’s ATPase activity in a pulse chase experiment with a 60 s second
quench delay versus an acid quench experiment under the same reaction conditions.
Figure 3.10b zooms in on the pre-steady-state region of the time course. The experiments
were performed in both the presence and absence of S2 peptide which stimulates only the
steady-state turnover. The burst amplitudes and rates were comparable in the presence
and absence of peptide for the pulse-chase and acid quench experiments respectively.
Furthermore, the burst amplitude in the pulse chase experiments approximately doubles
the acid quench amplitudes. Because the pulse chase burst amplitudes are stoichiometric
77
ACID QUENCH
PULSE CHASE
Lon ≥ 5 µM
Lon ≥ 5 µM
[α-32P]ATP ≤ 200 µM
[α-32P]ATP ≤ 200 µM
Reaction loop
t1 = 0 – 3 s
Reaction loop
t1 = 0 – 3 s
10 mM ATP
100 mM Mg(OAc)2
0.5 N formic acid
Exit loop
t2 = 0 s
Exit loop
t2 = 60 s
Collection tube
phenol chloroform
Collection tube
0.5 N formic acid
Scheme 3.4 – Illustration of the difference between an acid quench and pulse chase
Rapid quench experiment. The pulse chase experiment incorporates a 60 s second
quench delay following the quench with unlabeled Mg:ATP. This allows all of the
labeled ATP bound during t1 to age for 60 s before the enzyme was denatured with acid.
78
a
12.5
2
10
1.5
7.5
1
[ADP], µM
[ADP] / active site ( µM)
2.5
5
2.5
0.5
0
0
0.5
1
1.5
2
2.5
3
3.5
[ADP] / active site ( µM)
b
0.7
3.5
0.6
3
0.5
2.5
0.4
2
0.3
1.5
0.2
1
0.1
0.5
[ADP], µM
tim e (s)
0
0
0.1
0.2
0.3
0.4
time (s)
Figure 3.10 – Pre-steady-state ATPase activity of E. coli Lon protease as determined by
a pulse-chase and acid-quench rapid quench experiment. All experiments contained 5
µM Lon, 100 µM [α – 32P]ATP. a. (○) acid-quench + 500 µM S2 peptide (□) acidquench intrinsic (●) pulse-chase + 500 µM S2 peptide and (■) pulse-chase intrinsic. The
data from 600 ms to 3 s was best fit using a linear equation resulting in the steady-state
rate constants of 0.65 s-1, 0.24 s-1, 0.58 s-1, and 0.23 s-1 b. Figure 3.8b zooms in on the
pre-steady-state region of the time courses. The data from 0 to 400 ms were best-fit with
a single exponential equation (solid black lines) to yield a burst amplitude in the presence
and absence of S2 peptide respectively of 0.41 + 0.03 µM and 0.38 + 0.02 µM and a kburst
of 6.5 + 1.4 s-1 and 7.6 + 1.3 s-1 for the acid-quench experiments. Similarly in the
presence and absence of S2 peptide respectively, a burst amplitude of 0.65 + 0.05 µM
and 0.54 + 0.03 µM and a kburst of 19 + 3.1 s-1 and 19 + 2.3 s-1 were yielded for the pulsechase experiments.
79
to the amount of Lon present, all the monomeric subunits are capable of hydrolyzing
ATP. Although the Lon monomer sequence contains only one ATP binding domain,
Menon and Goldberg have previously observed that two affinities for ATP are detectable
(38). The two affinities were attributed to the existence of high- and low-affinity ATPase
sites in Lon when it is assembled in its oligomeric form. In other words, when Lon
assembles into a hexamer, half of the the monomeric subunits bind ATP with highaffinity and half of the monomeric subunits bind ATP with a lower-affinity (Figure 3.11).
Why the monomers diplay differing behaviors with respect to ATP is unknown. It is
possible that the monomers are not assembled into the hexamer identically resulting in
structural differences among the subunits. Additionally the two ATPase sites in Lon
could alternate between the high- and low-affinity behaviors. However, this possibility
would look kinetically identical to the sequential model we propose below. Our
explanation for the decreased burst amplitude detected in the acid quench experiments is
that there is coordinated ATP hydrolysis between the nonequilvalent ATPase sites in Lon
(i.e. the high- and low-affinity sites). We hypothesized a sequential reaction model for
ATP hydrolysis to account for the data (Figure 3.11). Assuming that 50% of the Lon
monomeric subunits exhibit high-affinity ATPase sites, they would be saturated at 5 µM
ATP which is the lowest concentration of ATP studied (Figure 3.6). At these
concentrations Lon binds but does not hydrolyze ATP. However, as the concentration of
ATP increases the low-affinity sites are occupied whose full occupancy activates
hydrolysis at the high-affinity sites. This explained the independence of the burst rate
constant toward the concentration of ATP (Figure 3.7c) as well as the half-burst or halfsite reactivity (Figure 3.7b).
80
fast
binding
kburst
12
kcat
s-1
1 s-1
Low-affinity ATPase site
High-affinity ATPase site
ATP bound low-affinity site
ATP bound high-affinity site
ADP bound low-affinity site
ADP bound high-affinity site
Figure 3.11 – Proposed model for Lon ATP hydrolysis activity. Lon is represented as a
hexamer with half of the monomers diplaying high-affinity ATPase sites (squares) and
half of the monomers diplaying low-affinity ATPase sites (circles). The high- and lowaffinity sites display functional non-equilvalency. We propose the binding of ATP to the
low-affinity sites stimulates ATP hydrolysis at the high-affinity sites. The low-affinity
sites would hydrolyze ATP at a slower rate following the burst in ADP production from
the high-affinity sites.
81
Correlation of Pre-Steady-State ATP and Peptide Hydrolysis Activities. In conclusion, E.
coli Lon protease exhibits pre-steady-state burst kinetics in the hydrolysis of ATP which
is consistent with a step following hydrolysis being rate limiting for turnover. The highand low-affinity ATPase sites hydrolyze the first round of ATP at different rates, so the
burst amplitude in the acid quench experiments is reduced by half. The activity of the
two sites must be coordinated in order to achieve
optimal steady-state hydrolysis of ATP and thus optimal peptide degradation. E. coli Lon
protease was found to exhibit lag kinetics in the degradation of the model peptide
substrate S3 (77). Figure 3.12 shows the burst in ATP hydrolysis and the lag in peptide
degradation under identical reaction conditions. During this lag, fifty percent of the Lon
monomer hydrolyzes ATP with a burst rate of 11.3 + 3.3 s-1. This indicates that the
hydrolysis of peptide requires the build up of a reaction intermediate, which coincides
with the burst in ATP hydrolysis. Collectively, the results support the model by which
peptide hydrolysis is activated by ATP hydrolysis. The rate limiting step in ATP
hydrolysis seems to be coupled with the first round of peptide cleavage. This model in
which ATP hydrolysis occurs prior to peptide cleavage disagrees with previous models
which suggested that peptide hydrolysis occurs before ATP hydrolysis (1). The
discrepancy is most likely due to the differing peptide substrates. As our model substrate
is more kinetically similar to protein substrates (40, 53), we conclude that it is more
suitable for evaluating the ATP-dependent protease mechanism for Lon. The energy
generated from ATP hydrolysis could be used to facilitate the proposed peptide
translocation step or induce a conformational change in the oligomer that is needed for
82
peptide hydrolysis. Pre-steady-state studies discussed in later chapters clarify these
possibilities.
83
10
1.6
8
1.2
6
0.8
4
0.4
2
0
0.5
1
1.5
2
ATP or peptide hydrolyzed
(µM)
product / active site
(µM)
2.0
0
time (s)
Figure 3.12 – Comparison of pre-steady-state ATP hydrolysis and S3 degradation at
identical reaction conditions (5 µM Lon monomer, 100 µM ATP, 500 µM peptide). The
time course for peptide hydrolysis (○) was monitored by fluorescence stopped-flow
spectroscopy. The time course for ATP hydrolysis (♦) was determined by rapid acid
quenching. Adapted from Vineyard, et al. (77).
84
CHAPTER 4
SINGLE TURNOVER EXPERIMENTS ISOLATE THE ATPase ACTIVITY AT THE
HIGH-AFFINITY SITES IN ESCHERICHIA COLI LON PROTEASE
85
Introduction
In the previous chapter the pre-steady-state characterization of the ATPase
activity of E. coli Lon protease was discussed. Lon was found to display a half-burst in
ADP production during the first turnover of the hydrolysis reaction (11 s-1), while there
was a pre-steady-state lag in peptidase activity (1 s-1) confirming that ATP hydrolysis
occurs prior to peptide cleavage. Because Lon is a homooligomer containing a single
ATPase domain per monomeric subunit, the half-burst detected in the pre-steady-state
ATPase activity was unusual. I demonstrated that the reduction in burst amplitude was
not a result of the inability of Lon to bind nucleotide or a contaminating population of
inactive enzyme. The pulse-chase experiments discussed in Chapter 3 resulted in a full
burst of ADP production suggesting that the high- and low-affinity ATPase sites
hydrolyze ATP at different rates. Therefore, the half burst in ATPase activity was
proposed to be a result of functional nonequivalency in the two ATPase sites. There is
precedent in other enzyme systems of two ATPase sites of differing affinities displaying
differing activities. The accepted model for p-glycoprotein is that ATP hydrolysis
alternates at the two nucleotide binding domains (85-88). The Na+/K+-ATPase has a
high- and low-affinity ATPase site which are proposed to function cooperatively during
transport (89, 90). Furthermore, the Rep helicase has two ATPase sites, of which only
one displays burst kinetics in ATP hydrolysis (91). Based upon the kinetic data obtained
in the study of the ATP-dependent degradation of the model peptide substrate (S3) by
Lon, we proposed a reaction model by which the nonequivalent ATPase sites function
cooperatively to maximize the efficiency of peptide cleavage (77). The model predicted
that ATP hydrolysis will occur with a burst rate constant of ~12 s-1 at the high-affinity
86
sites of Lon only when the low-affinity sites are occupied by ATP. This model would
account for the lack of dependency of the burst rate constant on the concentration of ATP
because the the first turnover of ATP hydrolysis is dependent on the binding of ATP to
the low-affinity sites (Figure 3.7c). Furthermore, optimal peptide hydrolysis is obtained
through the coordinated binding of ATP to the low affinity sites and hydrolysis at the
high-affinity sites. Therefore, the goal of this chapter is to describe the experiments used
to test this hypothesis. The experiments were performed under single turnover conditions
where the concentration of Lon is in excess of the concentration of ATP. Using single
turnover conditions, the concentrations of reactants can be manipulated to selectively
measure ATP hydrolysis at the high-affinity sites.
All of the pre-steady-state experiments described in Chapter 3 were performed
under pseudo-first-order conditions; which means that the concentration of ATP was in
excess over the concentration of Lon. Under these conditions ATP is bound at both the
high- and low-affinity (Kd, high-affinity < 1 µM, Kd, low-affinity = 10 µM (38)). Therefore, the
resulting ATP hydrolysis activity reflected both of the ATPase sites upon which the
reaction model was based. As such, the functional roles of the high- and low-affinity
ATPase sites could not be examined and the validity of the model could not be
thoroughly tested. In order to selectively examine the high-affinity ATPase sites a Kd
value for the high-affinity ATPase site needed to be determined. To this end, I
engineered a double filter binding assay to probe the binding affinity of ATP at the highaffinity site. This procedure was based on the filter binding assay described in Chapter 3
as well as assays described in literature (63, 92, 93). In order to selectively measure the
ATPase activity of the high-affinity sites, I utilized single turnover experiments. In these
87
experiments the concentration of monomeric enzyme (6 µM) is in excess of the
concentration of ATP (500 nM). Thus only the high-affinity ATPase sites should be
occupied by ATP, and the resulting rate constant for ATP hydrolysis assigned to the
high-affinity site ATPase activity. The experiments were performed in both the presence
and absence of peptide and protein substrate to account for their effect on ATP hydrolysis
at the high affinity sites. Based on its Kd (500 nM), few of the high-affinity sites would
be occupied under the limiting single turnover experimental conditions described above.
In order to occupy more high-affinity sites with ATP the concentration was increased to
~6 µM which is stoichiometric to the amount of enzyme present in the reaction. Scheme
4.1 shows the expected ATP occupancy of the enzyme forms under the various
conditions used. In Scheme 4.1, Lon is represented as a hexamer based on recent partial
crystal structures (31-33). The monomers displaying high- and low-affinity ATPase sites
in the hexamer are represented as squares and circles respectively. The occupancy of a
site by ATP is represented by the change in color from gray to yellow.
By performing the single turnover kinetic experiments under varying levels of
ATP, I was able to show that Lon exhibits two distinct kinetic behaviors in its ATPase
activity, with optimal peptide hydrolysis occurring upon full occupancy of ATP at both of
the sites. The high-affinity sites hydrolyze ATP very slowly (0.01 s-1) compared to the
low-affinity sites (12 s-1). Furthermore the ATP hydrolysis activity of the high-affinity
site is seemingly independent of the low-affinity site. Because multiple rounds of peptide
hydrolysis occur under conditions of limiting ATP, the two activities were shown to not
be stoichiometrically linked. Collectively, the kinetic data obtained using the single
88
No
ATP
Limiting
(500nM ATP)
Stoichiometric
(6µM ATP)
Saturating
(100µM ATP)
6µM Lon
A
C
B
D
Low-affinity ATPase site
High-affinity ATPase site
ATP bound low-affinity site
ATP bound high-affinity site
Scheme 4.1 – Enzyme Forms Associated with Various Concentrations of ATPa
a
Form A is a enzyme containing two different sets of ATPase sites that are represented
by gray squares and circles. Form B is formed under single-turnover when only 500 nM
ATP is present. The occupancy of ATP to an enzyme subunit is illustrated by the change
in color from gray to yellow. Form C represents the enzyme form where only the tight
sites are occupied by ATP. Form D represents an enzyme form where both the tight and
weak sites are saturated with ATP.
89
turnover experimental approach allowed for the revision of the previously proposed
reaction model and was published in Biochemistry (94).
90
Materials and Methods
Materials. ATP was purchased from Sigma whereas [α-32P] ATP was purchased from
Perkin Elmer or ICN Biomedical. Tris, HEPES and PEI cellulose TLC plates were
purchased from Fisher.
General Methods. Protein purification procedures were performed as described
previously (78). All enzyme concentrations were reported as Lon monomer
concentrations. All reagents are reported as final concentrations, and experiments were
performed at 37 °C unless otherwise indicated.
Double Filter Binding Assay. For the high affinity ATP site binding experiment, fifty
nanomolar [α32P]ATP was mixed with 0.005-6 µM Lon (92) in 15 µL of 50 mM HEPES
pH 8.0, 5 mM Mg(OAc)2, 75 mM KOAc, 2 mM DTT. Three microliters of the reactions
(performed in triplicate) were spotted onto a piece of nitrocellulose mounted onto a dotblot apparatus (BioRad) with a piece of Immobilon Ny+ below as described elsewhere
(63, 93). All reactions were performed at least in triplicate. Each spot was washed with
10 µL of cold reaction buffer two times. The radioactive counts at each spot were
quantified by PhosphorImaging using the Packard Cyclone storage phosphor system.
The concentration of bound was determined according to equation 1,


NCdlu
 * [α 32 P − ATP ]
[bound ] = 
+
 NCdlu + NY dlu 
(1)
where NCdlu are the radioactive counts on the nitrocellulose membrane and NY+dlu are the
radioactive counts on the Immobilon Ny+ membrane. The binding parameters were
determined by fitting the data with equation 2 using the nonlinear regression analysis
program Prism (GraphPad) software version 4.
91
[ RL] =
([ R] + [ L] + K d ) − ([ R] + [ L] + K d )2 − 4[ R][ L]
(2)
2[ L]
where [L] is the concentration of α32P-ATP, [R] is the concentration of Lon, [RL] is the
concentration of α32P-ATP bound to Lon, and Kd is the equilibrium dissociation constant
for ATP bound at the high affinity site.
When both the high- and low-affinity sites were probed the experiment was performed as
described above except that the concentration of Lon was varied from 5 nM – 30 µM, and
the data was fit with equation 3 using the nonlinear regression analysis program Prism
(GraphPad) software version 4.
[ RL] =
([ R] + [ L] + K d ) − ([ R] + [ L] + K d ,1 )2 − 4[ R][ L]
2[ R]
+
Bmax, 2 [ R]
K d , 2 + [ R]
(3)
where [L] is the concentration of α32P[ATP], [R] is the concentration of Lon, [RL] is the
concentration of α32P-ATP bound to Lon, Kd,1 is the equilibrium dissociation constant for
ATP bound at the high-affinity site, Bmax is the maximal bound complex detected, and
Kd,2 is the equilibrium dissociation constant for ATP bound at the low-affinity site.
Single Turnover ATPase Assays. Single turnover data for ATP hydrolysis was measured
as described elsewhere (55), and all reactions were performed at least in triplicate.
Briefly, for the ATPase measurements, each reaction mixture (70 µL) contained 50 mM
HEPES (pH 8.0), 75 mM KOAc, 5 mM Mg(OAc)2, 5 mM DTT, and 5 or 6 µM Lon
monomer. For the peptide-stimulated ATPase reactions, 500 µM peptide substrate (S2)
was added to each reaction mixture, and the reactions were initiated by the addition of
[α32P]ATP. Subsequently, 5 µL aliquots were quenched in 10 µL of 0.5 N formic acid at
twelve time points (from 0 to 15 min). A 3 µL aliquot of the reaction was spotted directly
92
onto a PEI-cellulose TLC plate (10 cm x 20 cm) and the plate developed in 0.75 M
potassium phosphate buffer (pH 3.4). Radiolabeled ADP was then quantified using the
Packard Cyclone storage phosphor screen Phosphor imager purchased from Perkin-Elmer
Life Science. To compensate for slight variations in spotting volume, the concentration of
the ADP product obtained at each time point was corrected using an internal reference as
shown in eq 4.

ADPdlu
[ ADP] = 
 ATPdlu + ADPdlu

 × [ ATP ]

(4)
All assays were performed at least in triplicate and the kinetic parameters were
determined by fitting the time course data with a single exponential equation 5 using the
nonlinear regression program Prism (Graphpad) software version 4.
Y = A * exp − k obs t + C
(5)
where t is time in seconds, Y is [ADP] in µM, A is the amplitude in µM, kobs is the
observed rate constant in s-1, and C is the end point.
Chemical Quench ATPase Activity Assays. The acid quenched time courses for ATP
hydrolysis were measured using a rapid chemical quench-flow instrument from KinTek
Corporation as described by Vineyard et al (77). All solutions were made in 50 mM
HEPES buffer pH 8.0, 5 mM DTT, 5 mM Mg(OAc)2, 75 mM KOAc. A 15 µL buffered
solution of 6 µM Lon monomer or 6 µM Lon pre-incubated with 6 µM ATP , with and
without 500 µM S2 or 20 µM casein, was rapidly mixed with a 15 µL buffered solution
of 100 µM ATP containing 0.01% of [α-32P]ATP at 37°C for varying times (0-3 s). The
reactions were quenched with 0.5N formic acid and then extracted with 200 µL of
phenol/chloroform/isoamyl alcohol pH 6.7 (25:24:1). A 3 µL aliquot of the aqueous
93
solution was spotted directly onto a PEI-cellulose TLC plate, and treated as above. All
assays were performed at least in triplicate and the average of those traces used for data
analysis. The burst amplitudes and burst rates were determined by fitting the kobs data
from 0 to 400 ms with equation 6.
Y = A * exp − k burst t + C
(6)
where t is time in seconds, Y is [ADP] in µM, A is the burst amplitude in µM, kburst is the
burst rate constant in s-1, and C is the end point. The observed steady state rate constants
(kss,ATP) were determined by fitting the data from 600 ms to 3 s with the linear function,
Y= mX +C, where X is time, Y is [ADP] / [Lon], m is the observed steady state rate
constant in s-1, and C is the y-intercept.
Data fitting was accomplished using the
nonlinear regression program Prism (GraphPad) software version 4.
Tryptic Digestions. Tryptic digest reactions in mixtures containing 6 µM Lon, 50 mM
HEPES (pH 8.0), 5 mM magnesium acetate, 2 mM DTT, + 500 µM S2 peptide, and
either 1 mM ATP, 6 µM ATP, or 500 nM ATP were started by the addition of 1/50 (w/w)
TPCK (N-p-tosyl-L-phenylalanine chloromethyl ketone)-treated trypsin with respect to
Lon. At 0, 2, 4, 20, and 40 min, a 3 µL reaction aliquot was quenched in 3 µg of soybean
trypsin inhibitor (SBTI) followed by boiling. The quenched reactions were then resolved
by 12.5% SDS-PAGE analysis and visualized with Coomassie brilliant blue.
94
Results and Discussion
The initial goal of the experiments presented in this chapter was to test the
hypothesis that ATP hydrolysis at the high-affinity sites is stimulated by ATP hydrolysis
at the low-affinity sites. According to this model no hydrolysis of ATP would occur at
the high-affinity sites until the low-affinity sites are occupied with ATP. However, the
experimental results presented in this chapter contradict those expected as the very
slow/negligible hydrolysis at the the high-affinity sites (0.01 s-1) was unaffected by
subsequent occupation of the low-affinty sites by ATP (94). In fact, in light of the
experiments discussed in this chapter, the pre-steady-state burst activity described in the
previous chapter was found to be attributed to the first turnover of ATP hydrolysis at the
low-affinity sites, rather than a stimulation of activity at the high-affinity sites. The
single-turnover experiments described in this chapter were designed to first measure
high-affinity ATPase activity, to account for the effect of low-affinity ATP binding and
hydrolysis on high-affinity ATPase activity, and finally to determine if high-affinity
ATPase activity alone was sufficient to support Lon peptidase activity.
Double FilterBinding Assays to Assess Kd Values of High- and Low-Affinity ATPase
Sites. In order to isolate and fully characterize the ATPase activity at the high-affinity
sites using single turnover kinetic experiments, an accurate value for the Kd high-affinity was
needed. The Kd for the low-affinity sites has been reported to be 10 µM, and the Kd for
the high-affinity sites was estimated to be less than 1 µM, but was not determined more
accurately (38). Once an accurate Kd-high-affinity value is known, the level of ATP
occupancy at the high- and low-affinity sites can be manipulated by adjusting the
concentration of ATP (Scheme 4.1). In order to determine Kd, high-affinity, I modified the
95
filter binding assay adapted from the protocol of Gilbert and Mackey, discussed in
Chapter 3, to a double filter method similar to one used by Wong and Lohman to probe
protein – nucleic acid interactions (63, 93). In this method a nitrocellulose membrane is
still used to bind the Lon:[α32P]ATP complex while the unbound [α32P]ATP is captured
during the washes on the positively charged Immobilon Ny+ nylon membrane placed
directly underneath the nitrocellulose. The half life of the Lon:ATPhigh-affinity is calculated
to be approximately 4 s at 37 °C. Therefore, to prevent the dissociation of the enzyme
nucleotide complex as well as hydrolysis of the nucleotide, it was important in this new
method to work very quickly and for everything to be kept cold to reduce the off rate of
ATP. The inclusion of a small amount of Tween 20 detergent in the cold wash buffer
(0.03%) also helped to reduce nonspecific binding interactions of the unbound nucleotide
with the membrane as well as nonspecific protein nucleotide interactions. Control
experiments were performed to ensure that no nonenzymatic [α-32P]ATP hydrolysis was
occurring under the reaction conditions. When determining a Kd, the amount of substrate
is typically varied at a constant level of enzyme which is at least five times lower than the
expected Kd (72). However, as the level of radioactive substrate is increased, the amount
of background signal generated interferes with the quality of the data. Therefore, I
adapted the protocol of Jia et al. and instead varied the amount of Lon at a constant level
of [α32P]ATP which eliminated the background interference (92). With all of these
modifications incorporated in the filter binding assay, 50 nM [α-32P]ATP was incubated
with varying amounts of Lon and the resulting Lon/[α-32P]ATP complex was
immobilized onto the nitrocellulose membrane, whereas unbound [α32P]ATP was trapped
by the positively charged nylon membrane place directly below the nitrocellulose filter.
96
A binding isotherm of the high-affinity ATPase site in Lon was generated by quantifying
the amount of 32P immobilized onto the nitrocellulose versus the nylon membrane as
described in Materials and Methods. Figure 4.1a shows that ATP binds to the highaffinity site with a Kd value of 0.52 + 0.10 µM. This value is consistent with Menon and
Goldberg’s reported value of Kd, high-affinity < 1 µM (38).
The Kd value reported by Menon and Goldberg for the low-affinity ATPase site
was 10 µM (38). I attempted using the double filter binding assay to detect both the lowaffinity Kd value as well as the high-affinity Kd value. The hypothesis was that I could
vary the amount of enzyme in a wide enough range to cover both affinities (5 nM – 30
µM) and fit the resulting curve with a two site binding isotherm (eq 3) in order to obtain
the two Kd values. As shown in Figure 4.1b, when this experiment was performed the
resulting data never reflected the appropriate values for either the high- or low-affinity
Kd. Most likely this is because the double filter binding method for verifying the lowaffinity Kd value is not ideal because the interaction of enzyme and nucleotide is not
strong enough to withstand the duration of the assay. If the half-life of the complex is
calculated using the value for the off rate of ATP from the low-affinity site (Chapter 6, ~
7 s-1), the value is less than one second. This makes the filter-binding assay an
inappropriate method for the determination of the low-affinity Kd value. Instead a
continuous method such as fluorescence spectroscopy would need to be performed. The
binding to this site has previously been determined under our reaction conditions using
steady-state kinetic methods (40), and the resulting affinity agreed with the published
value of 10 µM. Therefore, confirmation of the Kd value for the low-affinity site was not
pursued.
97
a
[bound], µM
0.04
0.03
Kd = 0.52 + 0.10 µM
0.02
0.01
0.00
0
1
2
3
4
5
6
7
[Lon], µM
b
0.05
[bound], µM
0.04
Kd, 1 ~ 0
Kd, 2 = 1.0 + 0.2 µM
0.03
0.02
0.01
0.00
0
10
20
30
40
[Lon], µM
Figure 4.1 – (a) Determining the Kd for the high-affinity ATPase site in E. coli Lon using
an adapted filter-binding assay. To monitor the binding of [α-32P]ATP to only the highaffinity site, various concentrations of Lon (5 nM – 6 µM) were incubated with 50 nM
[α32P]ATP at 4 °C. The amount of Lon/[α-32P]ATP was quantified by PhosphorImaging
of the nitrocellulose membrane, and the free [α-32P]ATP was quantified by
PhosphorImaging of the positively charged Immobilon Ny+. The amount of (bound)
complex (▲) was calculated as described in the Materials and Methods, and the
generated data were fit using a binding isotherm (eq 2). The resulting Kd value was 0.52
+ 0.10 µM for the high-affinity site. (b) Various concentrations of Lon (5 nM – 30 µM)
were incubated with 50 nM [α32P]ATP at 4 °C and quantified as in (a). The amount of
(bound) complex (■) was calculated as described in Materials and Methods, and the
generated data were fit using a two-site binding isotherm (eq 3).
98
Single Turnover Experiments Monitor ATP Hydrolysis at the High-Affinty ATPase Sites.
Single turnover experiments drive the formation of the E:S complex because the
concentration of enzyme is in excess of the substrate and only look at one turnover of a
reaction because no excess substrate is available. Because we wanted to selectively drive
the formation of Lon:ATPhigh-affinity and not Lon:ATPhigh- and low-affinity the Kd value for the
high-affinity ATPase site was confirmed. Single turnover kinetic experiments were then
employed to examine the activity of only the high-affinity ATPase site using the Kd, highaffinity
value as basis for the concentration of ATP in the reaction. By manipulating the
concentration of the enzyme (5 µM) such that it is in excess of the substrate (500 nM) the
high-affinity site could be selectively occupied by ATP (Kd = 0.52 µM, Figure 4.1a).
This enzyme form is shown as enzyme form B in Scheme 4.1. In these experiments all of
the available nucleotide (500 nM) should be bound and hydrolyzed over the course of the
reaction. Initially I performed this experiment using the rapid quench so that millisecond
time points could be collected. To this end increasing amounts of Lon (0 – 10 µM) was
pre-incubated with 500 µM S2 peptide or 10 µM casein and rapidly mixed with 500 nM
[α-32P]ATP prior to quenching with 0.5 N formic acid at time points from 0 – 10 s.
Figure 4.2 shows representative data in the presence of S2 peptide. No [α-32P]ADP
production is detected over background until 6 s, suggesting that ATP hydrolysis at the
high-affinity sites was occurring on a much slower time scale than the burst detected in
chapter 3 (detectable 0 – 1 s). Therefore, the experimental set up was adjusted to monitor
the reaction over a longer time scale. In order to accomplish this, increasing amounts of
Lon (5 – 10 µM) in the presence and absence of 500 µM S2 peptide were mixed with 500
nM [α-32P]ATP and quenched in formic acid at time points from 0 – 20 minutes. Figure
99
0.06
0.05
[ADP], µM
0.04
0.03
0.02
0.01
0
0
2
4
6
8
10
12
time (s)
Figure 4.2 – Single turnover experiment to detect ATP hydrolysis at the high-affinity
site. Lon was increased from (0 – 10 µM) in the presence of 500 µM S2 peptide and
rapidly mixed with 500 nM α-32P[ATP] in an attempt to detect hydrolysis at only the
high-affinity site. (○) 0 µM Lon (■) 2 µM Lon (♦) 7 µM Lon (▲) 10 µM Lon No
hydrolysis was detected over background until 8 s.
100
4.3 shows the single turnover experiment where 5 µM Lon in the presence and absence of
500 µM S2 peptide hydrolyzes 500 nM ATP presumably at the high-affinity sites
(Scheme 4.1, enzyme form B). The inset in Figure 4.3 shows the hydrolysis of ATP at the
high-affinity sites as the concentration of Lon is increased (5, 7, and 10 µM). The
observed rate constants range from 0.006 to 0.007 s-1 with a standard deviation of less
than 0.8%. Because the rate constants are identical at increasing concentrations of Lon,
the binding of ATP is not rate-limiting under the single-turnover reaction conditions
employed in the experiment. As demonstrated in Figure 4.3, the presence of S2 peptide
does not influence ATP hydrolysis at the high-affinity sites because the rate constant for
the reaction is 0.006 + 0.001 s-1 and 0.007 + 0.001 s-1 in the absence and presence of a
saturating amount (500 µM) S2 peptide, respectively. This is consistent with the
observation that the burst rate constant is also unaffected by the presence of S2 peptide
(Chapter 3) (77). The burst rate constant obtained under pseudo-first-order conditions
(Scheme 4.1, Enzyme form D) (kburst = 11.3 + 3.3 s-1), however, is much faster than the
rate constant obtained under single-turnover conditions (Scheme 4.1, Enzyme form B)
(0.007 + 0.001 s-1). The only difference between these experiments is the occupancy of
ATP at the low-affinity sites. This implies that, although one ATP binding site exists per
monomer, two functionally distinct ATPase sites are evident in the homo-oligomeric
form of Lon.
Hydrolysis of ATP at the High-Affinity Sites is Independent of Binding and Hydrolysis of
ATP at the Low Affinity Sites. If ATP binding and hydrolysis at the low-affinity site
affected ATP hydrolysis at the high-affinity sites one would expect a stimulation of the
101
0.4
0.3
0.4
[ADP], µM
[ADP], µ M
0.5
0.2
increasing
Lon
0.2
0.1
0.1
0.0
0.3
0.0
0
250
500
750
1000
1250
1500
time, (s)
0
250
500
750
1000
1250
1500
time, (s)
Figure 4.3 – Pre-steady-state time courses of ATPase activity of E. coli Lon under
single-turnover conditions. The “background” [α-32P]ADP determined in Figure 4.2 was
less than 0.05 µM and is insignificant in this time course. The time courses for ATP
hydrolysis at the high-affinity sites were determined by incubating 5 µM Lon with 500
nM [α-32P]ATP in the absence (■) and presence (▲) of 500 µM S2 peptide. The kobs
values were determined by fitting the time courses using a single-exponential equation as
described in Materials and Methods, yielding observed rate constants of 0.006 + 0.001
and 0.007 + 0.001 s-1 in the absence and presence of the S2 peptide, respectively. The
inset shows time courses for 500 nM [α-32P]ATP hydrolysis at the high-affinity sites in
the presence of 500 µM S2 peptide at increasing concentrations of Lon: 5 µM (▲), 7 µM
(♦), and 10 µM (□). The kobs values were determined as described above yielding
observed rate constants of 0.007 + 0.001, 0.007 + 0.001, and 0.006 + 0.001 s-1,
respectively.
102
high-affinity ATPase rate constant as the low-affinity sites are occupied with ATP. In
order to test this hypothesis the [ATP]:[Lon] ratio was incrementally increased until the
amounts were stoichiometric. To this end varying amounts of ATP in 1 µM increments
from 1 – 6 µM were incubated with 6 µM Lon in the presence and absence of 500 µM S2
peptide and the reactionsquenched with acid at reaction times varying from 0 to 20
minutes. The time courses were fit with a single exponential equation to extract the
observed rate constants which were then plotted versus the ATP:Lon ratio as shown in
Figure 4.4. As discerned in Figure 4.4, the rate constants remain relatively constant
(0.006 – 0.02 s-1) indicating there is no marked increase in the high-affinity rate of ATP
hydrolysis as the ATP:Lon ratio increases. However, the rate constants in the presence of
saturating (500 µM) S2 peptide are consistently higher than in the absence of S2
suggesting that peptide may be stimulating the high-affinity ATPase activity. Because
there is no stimulation in the high-affinity site hydrolysis rate constant as the low-affinity
sites are occupied with ATP, the hydrolysis activity of the high-affinity site is concluded
to be independent of ATP occupation at the low-affinity sites.
To further investigate the hypothesis that the high-affinity site ATPase activity is
independent of ATP occupation at the low-affinity sites further single-turnover
experiments were performed. Typically a substrate concentration is considered saturating
if it is ten times that of the Kd (72). As the Kd for ATP at the high-affinity sites is 0.52 +
0.10 µM, the single-turnover experimental conditions employed above (5 µM Lon, 500
nM ATP) were not sufficient to saturate those sites. To detect the full effect of ATP
hydrolysis when the high-affinity sites were fully occupied, the concentration of [α32
P]ATP was raised to approximately 10-fold excess of the Kd (6 µM), which is
103
0.02
k
obs
-1
(s )
0.015
0.01
0.005
0
0.2
0.4
0.6
0.8
1
1.2
ATP:Lon ratio
Figure 4.4 – The observed rate constants for ATP hydrolysis at the high-affinity sites in
the presence (■) and absence (●) of 500 µM S2 peptide plotted versus the ATP:Lon ratio.
No marked stimulation of the rate constant for high-affinity site ATP hydrolysis is
observed as the low-affinity sites are filled suggesting that the functionally distinct
ATPase activities are independent of one another.
104
stoichiometric to the amount of Lon in the reaction. Shown as enzyme form C in Scheme
4.1, under these conditions, it is assumed that the high-affinity sites are saturated and the
low-affinity sites are not bound to ATP. The experiment shown in Figure 4.5
demonstrates the effect on high-affinity site ATP hydrolysis when the low-affinity sites
are saturated with unlabeled nucleotide one half-life into the reaction. In this experiment
ATP hydrolysis was measured at the high-affinity sites under stoichiometric [α32
P]ATP/Lon conditions (enzyme form C in Scheme 1), while saturating (100 µM)
unlabeled ATP was subsequently added or chased to occupy the low-affinity sites 1
minute into the reaction with ATP (enzyme form D in Scheme 1). This allows for the
hydrolysis at the high-affinity sites to be measured for the first half-life and then the
effect of nucleotide occupation at the low-affinity sites on the high-affinity hydrolysis
should be evident. The rate constants for the various experiments are summarized in
Table 4.1. The experiments were always performed in both the absence (intrinsic) and
presence (S2 stimulated) of S2 peptide to account for the effects of any coupling between
the ATPase and peptidase activities. Control experiments were also performed using the
protein substrate casein, whose effect was comparable to S2 peptide (kobs, ATP = 0.02 +
0.01 s-1). To ensure that 60 s was an appropriate time to add saturating nucleotide, a
control experiment was performed where the saturating nucleotide was added at 10 s and
no difference was noted. As illustrated in Table 4.1, the hydrolysis at the high-affinity
sites was unaffected by occupation at the low-affinity sites 60 s into the reaction with
ATP, AMPPNP, or ADP. Because neither the nonhydrolyzable ATP analog (AMPPNP)
or the product inhibitor (ADP) affected the high-affinity ATPase activity, the high-
105
6
[ADP], µ M
5
4
3
2
1
0
0 100 200 300 400 500 600 700 800 9001000
time (s)
Figure 4.5 – Representative E. coli Lon time courses of ATP hydrolysis at the highaffinity sites. [α-32P]ATP (6 µM) was incubated with 6 µM monomeric Lon in the
absence (■) or presence (▲) of 500 µM S2 peptide and quenched with acid at varying
time points. To see the effect of nucleotide occupation at the low-affinity sites on the
high-affinity ATPase activity, 100 µM ATP was added at 1 half-life (60 s) into the
reaction in both the absence (▼) and presence (♦) of 500 µM S2 peptide to saturate the
low-affinity ATPase sites. The time of addition of the 100 µM ATP in traces ▼ and ♦ is
indicated by the arrow. The kobs, ATP is the observed rate constant obtained from the fit of
the data with a single exponential equation. The rate constants are summarized in Table
4.1. The time points reported are an average of at least three trials.
106
Table 4.1: Rate Constants Associated with High-Affinity Site ATPase Activity
Intrinsic
kobs,ATP (s-1)
S2 Stimulated
kobs,ATP (s-1)
Stoichiometric ATP
0.011 + 0.001
0.019 + 0.002
100µM ATP Chase
0.012 + 0.001
0.017 + 0.001
100µM AMPPNP Chase
0.010 + 0.001
0.014 + 0.001
100µM ADP Chase
0.015 + 0.001
0.017 + 0.001
107
affinity site ATP hydrolysis activity is independent of binding and/or hydrolysis at the
low-affinity sites.
Tryptic Digestion Reactions Probe the Nucleotide Dependent Conformational Change.
In light of the detection of functional nonequivalency in the ATPase activity in Lon we
questioned whether the ATP-dependent conformational change discussed in Chapter 2
could be assigned to a specific interaction between ATP with either the high- or lowaffinity ATPase sites in Lon. To address this issue, I subjected 6 µM Lon to limited
tryptic digestion in the presence of limiting (500 nM; enzyme form B Scheme 4.1),
stoichiometric (6 µM; enzyme form B Scheme 4.1), and excess (1 mM; enzyme form D
Scheme 4.1) amounts of ATP. By performing the digest reaction under these various
conditions we were able to monitor the effect on the stability of the adenine-specific
conformational change when one or both ATPase sites were occupied with ATP. On the
basis of the two Kd vales of ATP, we anticipated that only the high-affinity sites in Lon
were occupied by ATP in the first two cases. Figure 4.6 shows the Lon fragments
generated over increasing time in the presence of the S2 peptide under conditions of no
nucleotide (lanes 2-5), limiting (500 nM) ATP (lanes 6-9), stoichiometric (6 µM) ATP
(lanes 10-12), and saturating (1 mM) ATP (lanes 13-15). The digest pattern was identical
in the absence of the S2 peptide (data not shown). In accordance with what was
previously observed, limited tryptic digestion of Lon in the presence of ATP yields
fragments varying from 23 to 67 kDa. As indicated by these data, the 67 kDa fragment is
substantially stabilized only by full occupation of the low-affinity ATPase sites. Because
binding and hydrolysis of ATP at all ATPase sites are also necessary for optimal
108
No
ATP
500nM
ATP
1mM
ATP
6µM
ATP
kD
8
6
4
3
2
2
SBTI
Time
(min)
0
2
4
20
0
2
4
20
0
20
40
0
20
40
Figure 4.6 – Limited tryptic digestion of Lon in the presence of varying amounts of ATP.
Lon in the presence of 500 µM S2 peptide was digested with a limiting amount of trypsin
and quenched at the indicated times with SBTI as described in the Materials and
Methods. The first lane shows the molecular markers in kilodaltons (from top to
bottom): 172, 110, 79, 62, 48, 37, 24, and 19.
109
peptidase activity in Lon, a correlation between this adenine-specific conformational
change and accessibility for peptide cleavage could exist.
The Low-Affinity ATPase Sites Display a Pre-Steady-State Burst in ATP Hydrolysis.
Although the high-affinity ATPase activity has been isolated by utilizing single-turnover
experiments and the effect of subsequent occupation of the low-affinity sites has been
monitored, the low-affinity ATPase activity has not been isolated. As described in
Chapter 3 under pseudo-first-order conditions where the substrate concentration is in
excess of the enzyme concentration, Lon displays a pre-steady-state burst. However, the
burst activity was unusual in that it displayed half-site reactivity which was attributed to a
functional non-equivalency in the high- and low-affinity ATPase sites. The original
hypothesis was that the occupation of the low-affinity sites stimulated the high-affinity
sites which were responsible for the burst in ADP production. This explanation
accounted for the both the half-burst as well as the burst rate (kburst = 11.3 + 3.3 s-1)
lacking dependence on ATP concentration. However, in light of the results of the singleturnover experiments, the high-affinity sites are known to hydrolyze ATP very slowly
(kobs = 0.007 + 0.003 s-1) and independently of nucleotide binding or hydrolysis at the
low-affinity sites. Therefore, the pre-steady-state burst in ADP production is more likely
attributed to the low-affinity ATPase sites. Isolating the low-affinity sites proved to be
more difficult than the isolation of the high-affinity sites because they cannot be
selectively occupied by manipulating the concentration of enzyme and nucleotide. As an
alternative, I attempted to block the high-affinity sites with stoichiometric amounts of
unlabeled ADP (6 µM) and subsequently rapidly mix saturating amounts (100 µM) [α32
P]ATP to occupy the low-affinity sites. Figure 4.7 depicts this experiment using 6 µM
110
Lon pre-incubated with saturating (500 µM) S2 peptide, compared to an acid quench
experiment where the high-affinity sites are not blocked with ADP. The pre-incubation
of Lon with nucleotide was performed with both ATP, which was allowed time to
hydrolyze to ADP as well as with ADP directly in order to ensure that the hydrolysis
event at the high-affinity sites had no effect on the low-affinity ATPase activity. As
shown in Figure 4.6, when the high-affinity sites are occupied by 6 µM unlabeled ADP,
there is a decrease in the steady-state rate from 0.40 + 0.21 s-1 to 0.11 + 0.01 s-1.
However, the pre-steady-state burst rate and amplitude remain unaffected. This
experiment confirms the hypothesis that the low-affinity ATPase sites are responsible for
the pre-steady-state burst in ADP production. The steady-state rate when the highaffinity sites are blocked with ADP (0.11 + 0.01 s-1) is comparable to the intrinsic steadystate rate at 100 µM ATP (0.13 + 0.05 s-1) (55). This implies that the high-affinity sites
are susceptible to peptide stimulation. However, this experiment did not exclude the
possibility that the reduced steady-state rate was a result of ADP inhibition. The
experiments outlined below investigate whether the peptide induced stimulation of the
steady-state ATPase activity is a result of an interaction of the peptide with high-affinity
sites or whether the observed steady-state rate reduction (Figure 4.7) was due to ADP
inhibition which coincidentally resulted in a rate constant similar to intrinsic ATPase
activity.
ADP Inhibits the Steady-State ATPase Activity. In order to investigate this hypothesis
further, the amount of ADP pre-incubated with Lon was varied from (0 – 30 µM) and no
peptide was present. If the decreased steady-state rate results solely from the loss of
111
[ADP], µ M
9
8
7
6
5
4
3
2
1
0
0
1
2
3
4
time (s)
Figure 4.7 – E. coli Lon pre-steady-state chemical-quenched time courses of ATP
hydrolysis at the low-affinity sites. [α-32P]ATP (100 µM) was incubated with 6 µM
monomeric Lon (■) or 6 µM monomeric Lon pre-incubated with 6 µM ATP (▲) in the
presence of 500 µM S2 peptide. The pre-incubation of 6 µM Lon with 6 µM ATP
presumably resulted in 6 µM Lon/6 µM ADP, where the high-affinity ATPase sites were
saturated. The reactions were quenched with acid at the indicated times, and the
concentration of [α-32P]ADP generated in the reactions were determined by TLC
followed by PhosphorImaging. The time courses from 0 to 400 ms were fit with a single
exponential function resulting in a kburst for Lon (■) of 15.9 + 0.07 s-1 and a kburst for
Lon/ADP (▲) of 17.2 + 0.09 s-1. The kss, ATP were obtained by fitting the time courses
from 600 ms to 3 s with a linear function and dividing the slope by the [Lon] in the
reaction and were 0.40 + 0.02 s-1 for Lon (■) and 0.11 + 0.01 s-1 for Lon/ADP (▲). The
time points reported here are an average of at least three different trials.
112
peptide stimulation of the high-affinity sites, the time courses should be unaffected by
ADP as long as it is occupying only the high-affinity sites. Figure 4.8 shows the time
course for intrinsic ATP hydrolysis in the presence of increasing amounts of ADP. The
pre-steady-state portion of the time course is difficult to resolve indicating that some lowaffinity sites must also be blocked under the experimental conditions. This results in a
lower amount of signal and in turn more error in the pre-steady-state fits. Despite this
limitation, the time courses from 0 to 400 ms were fit with a single exponential equation
to yield the burst rate and amplitude. The time courses from 600 ms to 3 s were fit using
a linear function which yielded the steady-state rate. The steady-state rate constants are
obtained by dividing the steady-state rate by the concentration of enzyme present in the
reaction. The values for the burst amplitudes, burst rate constants, and steady-state rate
constants are summarized in Table 4.2. As the concentration of ADP increases, the fit of
the pre-steady-state time points with a single exponential function is very poor and
reflected in the errors of the amplitude and burst rate constants. However, there is a trend
reflected in Table 4.2 that the burst amplitude but not the burst rate decreases with
increasing concentrations of ADP. This trend can be explained because as more lowaffinity ATPase sites become occupied with increasing concentrations of ADP, less are
available for the burst in ADP production. However, the burst rate is unaffected because
what ATP remains bound at the low-affinity sites is hydrolyzed with the same burst rate
constant. The steady-state rates clearly show an inhibition trend. An IC50 plot of these
values was constructed and shown in Figure 4.9. The IC50 value extracted from this plot
is 4 + 1 µM, which reflects the concentration of ADP needed to reach one half the
maximal inhibitory affect under those experimental conditions. Because an ADP
113
30
25
[ADP], µM
20
15
10
5
0
0
0.5
1
1.5
2
2.5
3
3.5
time, (s)
Figure 4.8 – E. coli Lon pre-steady-state chemical-quenched time courses of ATP
hydrolysis at the low-affinity sites. [α-32P]ATP (100 µM) was incubated with 30 µM
monomeric Lon (○), 500 nM Lon pre-incubated with 30 µM ADP (□), 30 µM Lon preincubated with 1 µM ADP (◊), 30 µM Lon pre-incubated with 5 µM ADP (U), 30 µM
Lon pre-incubated with 15 µM ADP (x), and 30 µM Lon pre-incubated with 30 µM ADP
(+). The time courses from 0 to 400 ms were fit with a single exponential function and
the resulting burst rates and amplitudes summarized in Table 4.2. The kss,ATP values were
obtained by fitting the time courses from 600 ms to 3 s with a linear function and
dividing the slope by the [Lon] in the reaction (Table 4.2).
114
Table 4.2: Rate Constants Associated with ADP Inhibition of Intrinsic ATP
Hydrolysis
[ADP]
(µM)
Burst amplitude
(µM)
kburst
(s-1)
kss,ATP
(s-1)
0
21 + 3
0.7 + 0.1
0.29
0.5
20 + 2
0.8 + 0.1
0.27
1
13 + 3
0.8 + 0.3
0.17
5
14 + 8
0.4 + 0.3
0.15
15
2+1
33 + 19
0.08
30
7 + 10
0.4 + 0.9
0.02
115
1
0.6
ss
k /k
ss,0µM ADP
0.8
0.4
0.2
0
-5
0
5
10
15
20
25
30
35
[ADP], µM
Figure 4.9 – IC50 plot of steady-state rate constants associated with ATP hydrolysis at
increasing concentrations of ADP (0 – 30 µM) and 30 µM Lon. The resulting IC50 value
under these experimental conditions was 4 + 1 µM.
116
inhibitory effect was apparent in these experiments where no peptide was present, the
decreased steady-state rate in Figure 4.7 does not indicate that the high-affinity sites are
responsible for the peptide stimulation of the steady-state rate constant.
Therefore,
although the pre-steady-state burst in ADP production can be attributed to the lowaffinity sites the effect from the high-affinity sites on this hydrolysis has not been
isolated. The effect of the high-affinity sites on the hydrolysis of ATP at the low-affinity
sites will need to be determined using other methods i.e. the high-affinity sites will need
to be inactivated using other means such as mutagenesis or covalent inhibitors. However,
more information regarding what determines each Lon monomer to display a high- or
low-affinity ATPases site would be required to pursue this.
117
CHAPTER 5
(MDCC)- LABELED PHOSPHATE BINDING PROTEIN IS USED TO PROBE
PHOSPHATE RELEASE IN THE ATPase MECHANISM OF ESCHERICHIA
COLI LON PROTEASE
118
Introduction
Along with ADP, inorganic phosphate (Pi) is a product of ATP hydrolysis in Lon
protease. In order to fully characterize the ATPase mechanism of Lon, the rate constant
associated with Pi release needs to be measured. A variety of spectroscopic assays exist
to detect Pi including the malachite green assay described in Chapter 2, however, these
assays do not measure Pi in real time (76). Martin Webb and colleagues enhanced assays
for Pi by developing a rapid and sensitive fluorescent probe for Pi (95). They utilized the
affinity of phosphate binding protein (PBP) for Pi and introduced the fluorophore 7diethylamino-3-[[[(2-maleimidyla)ethyl]amino]carbonyl] coumarin (MDCC) in PBP
which is sensitive to Pi binding. I chose to utilize this assay for measuring Pi production
in the Lon system because, if Pi release were rate limiting in the Lon ATPase pathway,
the rate constant can be calculated from the data.
Phosphate binding protein (PBP) is the product of the phoS gene in E. coli which
is induced when the levels of Pi are low, is localized to the periplasmic space, and is
implicated in the transport of Pi (95-97). Crystallization studies of phosphate binding
protein have shown that the protein consists of a single polypeptide chain containing a
single tight binding site for Pi (98). Like other binding proteins such as arabinose binding
protein and maltodextrin binding protein, PBP undergoes a ligand induced
conformational change where a hinge motion closes the cleft around the ligand (99, 100).
Martin Webb and colleagues (National Institute for Medical Research) exploited this
conformational change by introducing the mutation A197C in PBP near the edge of the
cleft (95). The introduced cysteine was covalently labeled with the fluorophore MDCC
119
O
O
N
O
Coumarin
S
N
N
H
O
O
Ethylene
linker
Succinimide
Figure 5.1 – Structure of the MDCC-thiol adduct
120
R
(Figure 5.1), and the resulting fluorescent signal is sensitive to Pi binding. Presumably,
the increase in fluorescence detected upon binding of Pi is a result of changes in the
environment of the coumarin due to the conformational change. Thus, a probe was
created for Pi which can rapidly measure micromolar concentrations released from
enzymes in real time based on to the increase in fluorescence resulting from MDCC-PBP
binding Pi.
Previously, MDCC-PBP was used to probe phosphate release in the kinetic
mechanism of kinesin and myofibrillar ATPase (101, 102). The goal of my project was
to monitor the individual rate constants associated with ATP hydrolysis in the Lon
protease system among which include binding, hydrolysis, and product release (ADP &
Pi). ADP binds tighter to Lon than ATP and its release has been suggested to be rate
limiting in turnover because ADP does not support proteolysis (1). The release of
phosphate, however, is thought to be fast because Lon:32Pi was never recovered in
equilibrium binding studies (39). Therefore, MDCC-PBP could be used to continuously
monitor ATP hydrolysis activity in the pre-steady-state analogous to the rapid quench
studies. By comparing the discontinuous rapid quenched time course with the continuous
stopped flow MDCC-PBP assay, if the Lon:Pi intermediate was slow to dissociate, a
difference in the time courses should be apparent because the fluorescent signal is
determined by the dissociation of the Lon:Pi complex. If the hypothesis that Pi release is
fast was correct, then the time course monitored using MDCC-PBP should overlay the
rapid quench time course. However, if Pi release is slow, then a lag would likely be
detected in the pre-steady-state rather than a burst.
121
A PBP clone from the K-12 strain of E. coli was obtained (generous gift from Dr.
Susan Gilbert, University of Pittsburgh) in order to probe phosphate release in the Lon
system. However, we also cloned PBP from the DH5α strain of E. coli in order to
increase the yield and simplify the expression of phosphate binding protein for our
purposes. The overexpression, purification, fluorescent labeling, and characterization of
PBP will be described in detail in this chapter. Once functional MDCC-PBP was
obtained, it was used to probe the ATPase activity of E. coli Lon protease.
122
Materials and Methods
Materials. Nucleotides were purchased from Sigma or ICN Biomedical. PNPase and 7MEG were purchased from Sigma. MDCC was purchased from Molecular Probes.
Sephacryl S-100 was purchased from Amersham Bioscience. Cloning reagents were
purchased from Promega, New England BioLabs Inc., Invitrogen and USB Corporations.
Oligonucletoides (primers) were purchased from Integrated DNA Technologies Inc.
General Methods. All enzyme concentrations were reported as Lon or PBP monomer
concentrations. All reagents are reported as final concentrations. Unless otherwise stated
all experiments were performed at 37 °C.
Cloning and Expression of Phosphate Binding Protein (PBP). The phosphate binding
protein (PBP) gene with the attached phoS signal sequence was amplified from genomic
DNA of the DH5α strain of E. coli using the forward primer 5’GGAATTCCATATGAAAGTTATGCGTACC-3’ and the reverse primer 5’CCCAAGCTTTTATTAGTACAGCGG-3’. An A197C mutation was then introduced using
PCR primer site directed mutagenesis and the additional forward primer 5’GTTGAATATTGTTACGCGAAG-3’ and reverse primer 5’-CCTCGCGTAACAATATTCAAC-3’.
The resulting product was cloned into the HindIII and Nde1 sites of the pET-24c(+)
vector and the resulting plasmid named pHF019. This sub-cloned phosphate binding
protein from E. coli DH5α contained one residue (Y306F) which differed from the
originally cloned protein.
Fermenting PBP. The BL21 (DE3) glycerol stock of pHF019 was streaked on luria broth
(LB)/30 µg/mL kanamycin agar plate and incubated overnight at 37 °C to get single
colonies. A single colony was then used to inoculate a 100 mL 30 µg/mL kanamycin LB
123
liquid overnight culture (220 rpm, 37 °C). Six 1.5 L super broth (SB) liquid cultures (30
µg/mL kanamycin) were innoculated with 15 mL of overnight LB culture. The cultures
grew to OD600 = 1.5 in approximately five hours and were induce with 1 mM IPTG for 1
hour and 45 minutes. The osmotic lysis procedure was started immediately following
induction. The cells were pelleted at 4000 rpm, 25 °C for 10 minutes in the GS-3 rotor
and the supernatant discarded. The cell pellets were resuspended in 1.5 L total 10 mM
Tris pH 7.6/30 mM NaCl. The cells were again pelleted at 4000 rpm, 25 °C for 20
minutes in the GS-3 rotor and the supernatant discarded. Pre-weighed two 400 mL
centrifuge bottles. The cells were resuspended in 800 mL total of 10 mM Tris pH 7.6/30
mM NaCl, transferred to the pre-weighed bottles, and then pelleted at 5000 rpm, 25 °C
for 20 minutes in the GS-3 rotor. The pellets were weighed and the GSA rotor prechilled to 4°C. Each weighed pellet was resuspended in 50 mL of 33 mM Tris pH 7.6, 25
°C and transfered to a single GSA centrifuge bottle containing a stir bar. 100 mL of 40%
sucrose/0.1mM EDTA/33mM Tris-HCl pH 7.6 was added slowly while rapidly stirring
for 10 minutes at 25 °C. The cells were pelleted by spinning in GSA rotor at 10,000 rpm,
4 °C for 20 minutes and discard soup. The SA-600 rotor was pre-chilled to 4 °C. The
cell pellet was rapidly resuspended in 200 mL of ice cold 0.5 mM MgCl2 and stir for 15
minutes. The cell debris was pelleted in SA-600 rotor at 10,000 rpm using polypropylene
centrifuge tubes for 20 minutes at 4 °C. The supernatant containing PBP was poured into
a chilled beaker. The supernatant should be a faint purple color. A 12.5% SDS PAGE of
the supernatant and pellet was run to ensure that PBP is present. The supernatant was
then dialyzed in 4 L of 5 mM Tris pH 8, 4 °C for 1 hour, flash frozen in dry ice, and
stored at -80 °C until purification.
124
Purification of PBP. This purification procedure and labeling of C197 with MDCC are
described in detail in reference 52. I made modifications to this procedure which is
delineated below. A 75 mL bed volume Q-sepharose column was poured and
equilibrated in 5 mM Tris pH 8 (600 mL). The tubes of lysate were thawed at room
temperature and loaded onto the Q-sepharose column at 2.5 mL/min. PBP was
isocratically eluted from the column by washing with 5 mM Tris pH 8, 25 mM NaCl, 5
mL/min. The concentration of the protein was then determined according to equation 1.
[PBP ] = ( A280 − A−1320 )−1
(1)
60,880 M cm
The purified PBP (MW = 34453.8 g/mol) was dialyzed overnight in 4 L 20 mM Tris pH
8.0 concentrated to a volume of ~100 µM for labeling, and the bound phosphate was
stripped from PBP by adding 2 unit/mL PNPase + 1 mM 7-MEG and rocking at room
temperature for 4 hours. A fresh stock of 25 mM MDCC was made in DMF. The mop
system (0.5 unit/mL PNPase, 200 mM MEG) was utilized for 30 min to rid spin vanes of
excess Pi. While stirring continually with spin vane, MDCC was added to PBP in
increments over 30 min to reach a final MDCC concentration of 0.15 mM. At 30 min, 1
mM BME was added to quench the reaction and allowed to incubate for 5 min, and the
reaction was diluted to 10 mM Tris. The labeled protein was concentrated to an
appropriate volume for a gel filtration column and dialyzed overnight into 10 mM
HEPES pH 7.2. A 300 mL bed volume Sephadex S-300 column was poured and
equilibrated in 10 mM HEPES pH 7.2, 0.01% Tween 20. MDCC-PBP was loaded on
and eluted from the gel filtration column both to separate any excess MDCC as well as
PNPase from the MDCC-PBP. The concentration of MDCC-PBP was then determined at
125
A430nm using the extinction coefficient for MDCC (46,800 M-1s-1) because the A280nm is
affected by other proteins such as PNPase.
Checking Activity of MDCC-PBP. Emission scans of MDCC-PBP (excitation 425 nM,
slits 2 nM and 2 nM) were performed in the absence and presence of Pi. The reaction
contained 2 µM MDCC-PBP, 0.1 unit/mL PNPase, 150 µM MEG, 50 mM HEPES pH 8,
75 mM KOAc, 5 mM MgOAc2, 5 mM DTT, in the absence and presence of 100 µM Pi.
If the MDCC-PBP did not respond with an increase in fluorescence in the presence of Pi,
it was again “stripped” of any bound phosphate using the phosphate “mop” system (2
unit/mL PNPase, 1 mM 7-MEG for 2 hours mixing end over end). The labeled protein
was dialyzed overnight in 4 L of 10 mM HEPES pH 7.2 to eliminate the lower molecular
weight components of the mop system.
Steady-State ATPase Assays using MDCC-PBP. Steady-state velocity data for ATP was
measured using MDCC-PBP to detect inorganic phosphate (Pi) release following ATP
hydrolysis. Reactions contained 50 mM Tris at pH 8.0, 5 mM Mg(OAc)2, 2 mM DTT,
150 µM 7-methylguanosine (MEG), 0.05 unit/mL PNPase, 300 nM or 150 nM E. coli
Lon, + 500 µM S2 peptide, and 25 µM - 1 mM ATP. A calibration curve for MDCCPBP was generated using known concentrations of Pi. ATPase activity was monitored on
a Fluoromax 3 spectrofluorimeter (Horiba Group) where MDCC-PBP fluorescence was
excited at 425 nm and emitted at 465 nm. The velocity reactions were equilibrated at 37
° C for one minute and initiated with the addition of Lon. Initial velocities were
determined from plots of the amount of Pi released versus time. All assays were
performed at least in triplicate, and the kinetic parameters were determined by fitting the
126
averaged rate constant data with equation 2 using the nonlinear regression program
KaleidaGraph (Synergy) version 3.6.
k obs =
k cat [ATP ]
K m + [ATP ]
(2)
where kobs is the observed rate constant in s-1, kcat is the maximal rate in s-1, [ATP] is the
nucleotide concentration in µM, and Km is the Michaelis-Menten constant in µM.
Stopped Flow Experiments using MDCC-PBP.
Pre-steady-state experiments were
performed on a KinTek Stopped Flow controlled by the data collection software Stop
Flow version 7.50 β. The sample syringes were maintained at 37 ºC by a circulating
water bath. The sample syringes were also cleaned of Pi using the “mop” system (0.5
unit/mL PNPase, 150 µM 7-MEG). Syringe A contained 5 µM E. coli Lon monomer
with and without 20 µM casein, 5 mM Mg(OAc)2, 50 mM HEPES pH 8, 75 mM KOAc,
5 mM DTT, 150 µM 7-MEG, 0.05 unit/mL PNPase, and varying MDCC-PBP (25 -100
µM). Syringe B contained (25 - 200 µM) ATP, 5 mM Mg(OAc)2, 50 mM HEPES pH 8,
75 mM KOAc, and 5 mM DTT, 150 µM 7-MEG, 0.05 unit/mL PNPase, and varying
MDCC-PBP (25 -100 µM). ATP hydrolysis was detected by an increase in fluorescence
(excitation 430 nm emission 400 nM long pass cut off filter) resulting from rapid mixing
of the syringe contents in the sample cell. The data were a result of averaging at least
four traces. All experiments were performed at least in triplicate. Calibration curves
were generated by monitoring the increase in fluorescence as MDCC-PBP bound varying
known concentrations of Pi.
127
Results and Discussion
Cloning and Fermentation of Phosphate Binding Protein (PBP). The original plasmid
containing PBP from Martin Webb (National Institute of Medical Research, Mill Hill,
London) was generously given to us by Susan Gilbert (University of Pittsburg).
However, the available phosphate binding protein gene was cloned into a vector which
contained tetracycline resistance and was induced by rhamnose sugar (103, 104). In
order to increase the yield and simplify the expression of phosphate binding protein for
our purposes we amplified the phosphate binding protein gene containing the phoS signal
sequence from the genomic DNA of the DH5α strain of E. coli and introduced the
A197C mutation by PCR primer site directed mutagenesis. Once verified by DNA
sequencing the resulting gene was cloned into the pET-24c (+) and the plasmid named
pHF019 as described in Methods and Materials. The phosphate binding protein from E.
coli DH5α contained one residue (Y306F) which differed from the originally cloned E.
coli K-12 protein. The plasmid pHF019 was transformed into E. coli BL21 (DE3) for
over-expression, selected for with 30 µg/mL kanamycin, and an induction study
performed at varying OD600 and times with 1mM IPTG. Figure 5.2 clearly shows that
more PBP is produced at OD600 = 1.5 when induced for at least 90 minutes.
MDCC-PBP Displays an Increase in Fluorescence Upon Binding to Pi. PBP was
isolated by osmotic lysis, purified to homogeneity, and labeled with the MDCC
fluorophore at C197 as described in detail in Materials and Methods. One complication
that arose throughout the purification of both strains of PBP was extraneous Pi
contamination. This would result in no change in fluorescence upon the addition of Pi in
128
OD600 = 0.5
OD600 = 1.0
OD600 = 1.5
Time (min) 30 60 90 120 30 60 90 120 30 60 90 120
Figure 5.2 – SDS-PAGE visualized by Coomassie Brilliant Blue of pHF019 PBP
induction study. Lane 1 shows the molecular markers in kilodaltons (from top to
bottom): 183, 114, 81, 64, 50, 37, 26, 20. Lane 2 shows a MDCC-PBP standard.
129
an emission scan. Webb and colleagues developed a phosphate “mop” system consisting
of 150 µM 7-methylguanosine (7-MEG) and 0.05 units/mL purine nucleoside
phosphorylase (PNPase) which removes excess phosphate by converting it to ribose-1phosphate (Figure 5.3) (95, 96, 105). At these concentrations there was no competition
between MDCC-PBP and the phosphate “mop” system for Pi. This “mop” system was
used to wash the cuvettes as well as in the reaction mixtures. However, these “mop”
conditions were not stringent enough to rid MDCC-PBP of all Pi following purification
and labeling. Therefore, the more stringent conditions of 2 unit/mL PNPase, 1 mM 7MEG were employed to “strip” MDCC-PBP of extraneous phosphate. Subsequent to the
“stripping” of MDCC-PBP the protein was dialyzed to rid the lower molecular weight
components of the “mop” system (ie. 7-MEG) from the MDCC-PBP solution. Although
intact trimeric PNPase has a molecular weight of ~ 78 kDa and monomeric PBP has a
molecular weight of ~ 35 kDa, the two proteins proved very difficult to separate. As
shown in Figure 5.4, although the gel filtration column performed following the labeling
should have separated the two proteins, it failed to do so. Therefore an abundance of
PNPase is retained with PBP following the purification and labeling. As shown in Figure
5.5, despite the PNPase contamination, the purified labeled, and “stripped” protein
exhibits activity that is comparable to the PBP purified from the original cell strain (a
generous gift from Susan Gilbert, University of Pittsburg) developed by Webb and
colleagues (95). This emission scan was performed in Lon reaction buffer following
many trials with reagents of varying purity to reduce Pi contamination. Malachite green
(Chapter 2) was used to help determine the reagents with the highest amount of Pi
130
CH3
O-
+
N
N
HO
O
H
H
H
OH
H
OH
N
N
NH2
+
OH
Purine nucleoside
phosphorylase
(PNPase)
O
P
CH3
O
N
N
OH
HO
NH
N
NH2
O
O
+
H
H
H
OH
H
OH
P OH
O
OH
HO
Figure 5.3 – Depiction of the phosphate “mop” system consisting of PNPase using Pi to
convert 7-MEG to the nucleobase and ribose 1-phosphate.
Figure 5.4 – Lane 1 shows the molecular markers in kilodaltons (from top to bottom):
183, 114, 81, 64, 50, 37, 26, 20. Lane 2 shows a MDCC-PBP standard, and Lane 2
shows a PNPase standard. Lanes 4, 5, and 6 show increasing amounts (2, 4, 6 µL of 28.3
µM) of stripped MDCC-PBP that had been purified on a 300 mL bed volume Sephacryl
S-100 HR gel filtration column (fractionation range 1 X 103 – 1 X 105). Despite the fact
that all the know proteins fall within the fractionation range, no separation is obtained.
131
relative fluorescence
a
1 10
6
8 10
5
6 10
5
4 10
5
2 10
5
0
420
440
460
480
500
520
540
wavelength (nm)
b
6
2.5 10
relative fluorescence
2 10
6
6
1.5 10
1 10
6
5 10
5
0
420
440
460
480
500
520
540
wavelength (nm)
Figure 5.5 – Emission scan of 2 µM MDCC-PBP from (a) pHF019 or (b) original cell
strain in the presence (□) and absence (○) of 100 µM Pi. There is an approximate 3 fold
increase in fluorescence when MDCC-PBP binds to Pi.
132
contamination. The increase in fluorescence was greater when the scan was performed in
only water presumably due to less Pi contamination.
MDCC-PBP Steady-State Assay for Lon ATPase Activity. In order to determine that
MDCC-PBP was an acceptable probe for Pi release in the Lon system the steady-state
ATPase activity was monitored both in the absence and presence of 500 µM S2 peptide.
Because E. coli Lon is stored in 75 mM KPi to enhance its long term stability, Lon had to
be put through a gel filtration column to exchange it into a storage buffer lacking any Pi.
The phosphate free Lon displayed comparable activity to the Lon stored in phosphate.
For the steady-state ATPase assay, the concentration of MDCC-PBP first had to be varied
(25 µM – 250 µM) to ensure that the fluorescent signal wouldn’t saturate in the middle of
the reaction time course. At 125 nM Lon and 250 µM MDCC-PBP the time courses
showed an increase in fluorescence at 1 mM ATP in the presence and absence of 500 µM
S2 peptide as well as at 25 µM ATP in the absence of peptide. The rate of the time
courses doubled when the concentration of Lon was doubled, ensuring that the
fluorescent signal was a result of Lon hydrolyzing ATP. Practically speaking, these
controls would need to be performed each time a new batch of MDCC-PBP is purified.
As shown in Figure 5.6 a calibration curve for MDCC-PBP was the generated by
performing emission scans (excitation 425 nm) at increasing concentration of Pi (10 –
100 µM). The steady-state characterization using MDCC-PBP could then be performed.
The concentration of ATP was varied from 25 µM to 1 mM at 150 nM Lon in the
presence of 500 µM S2 peptide, and 300 nM Lon in the absence of peptide. Figure 5.7
shows the kcat/Km profile of the ATPase activity of Lon protease in the absence and
presence of peptide as determined using MDCC-PBP and [α-32P]ATP. Table 5.1
133
4
4.5 10
y = 6974.1 + 351.78x R= 0.99755
4 10
4
4
relative fluorescence
3.5 10
3 10
4
4
2.5 10
2 10
4
4
1.5 10
1 10
4
5000
0
20
40
60
80
100
120
[P ], µM
i
Figure 5.6 – Calibration curve of 250 µM MDCC-PBP in 50 mM HEPES pH 8, 5 mM
Mg(OAc)2, 75 mM KOAc, 5 mM DTT, 0.05 unit/mL PNPase, 150 µM MEG, and 1 mM
ATP.
134
2.5
1.5
k
obs
-1
(s )
2
1
0.5
0
0
200
400
600
800
1000
[ATP], µM
Figure 5.7 – Comparison of steady-state ATPase activity of E. coli Lon protease
determined by the MDCC-PBP assay (excitation 425 nm, emission 465 nm) in the
absence (○) and presence (●) of 500 µM S2 peptide and the [α-32P]ATP radioactive assay
in the absence (□) and presence (■) of 500 µM S2 peptide. The solid black line shows
the fit of the Michaelis Menten equation and the resulting parameters are summarized in
Table 5.1.
Table 5.1: Summary of ATPase Steady-State Kinetic Parameters from Comparison of
MDCC-PBP and [α-32P]ATP Assays
kcat, intrinsic (s-1)
Km,intrinsic (µM)
kcat,S2 stimulated (s-1)
Km,S2 stimualted (µM)
MDCC-PBP
0.53 + 0.05
40 + 16
2.4 + 0.1
57 + 10
[α-32P]ATP
0.26 + 0.01
46 + 6
1.4 + 0.1
82 + 10
135
summarizes the kinetic parameters obtained from fitting the plots shown in Figure 5.7
with the Michaelis-Menten equation. The kinetic parameters resulting from the two
methods are comparable, indicating that the MDCC-PBP system is acceptable for
monitoring steady-state ATP hydrolysis by E. coli Lon protease.
Probing for Pi Release from Lon. The stopped flow instrument was used to probe the
release of phosphate from Lon on a millisecond time scale. Prior to all stopped flow
experiments, the instrument was “mopped” of residual phosphate using 150 µM 7-MEG
and 0.05 units/mL (PNPase) for 15 minutes. To test the assay 5 µM Lon incubated with
10 µM casein or 500 µM S2 peptide was rapidly mixed with 300 µM ATP in the
presence of increasing amounts (25-100 µM) MDCC-PBP for 6 s. A linear time course
was observed for a period of time which would saturate presumably when the available
MDCC-PBP was all bound to Pi (Figure 5.8). 25 µM MDCC-PBP proved to be sufficient
for monitoring a Lon time course for 6 s. A linear calibration curve was generated
(Figure 5.9) using 25 µM MDCC-PBP. The calibration curve was generated under
identical buffer conditions as the Lon ATPase assay. The slope of the line was used to
convert relative fluorescence to the concentration of Pi in the ATPase time courses.
Figure 5.10 shows time courses of ATP hydrolysis by 5 µM Lon in the absence and
presence of 10 µM casein as detected using 25 µM MDCC-PBP. This figure
demonstrates that 25 µM MDCC-PBP proved to be sufficient to monitor both intrinsic
and protein stimulated ATPase time courses up to 6 s. However, the pre-steady-state
region of the time course (0 – 0.4 s) is affected by the presence of the phosphate “mop”
system. Figure 5.10 b zooms in on the pre-steady-state regions of the time courses.
From this figure it is clear that the fluorescent signal is affected in the initial portion of
136
2
relative fluorescence
1.5
1
0.5
0
0
2
4
6
8
10
time, (s)
Figure 5.8 – Fluorescent stopped-flow time courses (excitation 430 nm) of increasing
amounts of MDCC-PBP (V) 25 µM (PMT 513), (□) 50 µM (PMT 502), (○) 100 µM
(PMT 502) detection of Pi released from 5 µM Lon in the presence of 10 µM casein
hydrolyzing 300 µM ATP.
y = 2.2814 + 0.044083x R= 0.99949
relative fluorescence
4
3.6
3.2
2.8
2.4
0
10
20
30
40
50
[P ], µM
i
Figure 5.9 – Calibration curve of 25 µM MDCC-PBP using fluorescent stopped flow
(excitation 430 nm). A linear increase in fluorescence is detected with increasing
amounts of Pi.
137
a
10
8
i
[P ] / [Lon]
6
4
2
0
0
1
2
3
4
5
6
time, (s)
b
0.2
i
[P ] / [Lon]
0
-0.2
-0.4
-0.6
0
0.1
0.2
0.3
0.4
0.5
time, (s)
Figure 5.10 – (a) Detection of ATPase activity of 5 µM Lon protease in the absence (○,
□) and presence (x, ◊, V) of 10 µM casein at varying concentrations of ATP (○) 25 µM,
(□) 300 µM, (◊) 5 µM, (x) 25 µM, and (V) 300 µM using fluorescence stopped flow
(excitation 430 nM). The box indicates the region of the time course that is enlarged in
5.7b. (b) zoom in on first 500 ms of time course.
138
the time course (0 – 0.2 s) until enough Pi is generated from the Lon ATPase reaction to
out-compete the PNPase present in the reaction. Figure 5.11 confirms this hypothesis by
showing that the decrease in fluorescence in the pre-steady-state ATPase time courses
(~0-1 s) increases as increasing amounts of the “mop” system (PNPase and 7-MEG) are
added to the reaction. In order to ensure a population of responsive MDCC-PBP, the
“mop” system proved to be essential during the purification and labeling of the protein.
However, the presence of the Pi mop prevents the accurate monitoring of the pre-steadystate region of the ATPase time courses because it is competing with MDCC-PBP for the
Pi generated from the ATPase reaction. Therefore, the pre-steady-state ATPase activity
of Lon cannot be measured with MDCC-PBP. MDCC-PBP was also used to monitor
ATPase activity under single turnover conditions where the concentration of Lon (5 µM)
is in excess of the concentration of ATP (500 nM). However the competition for Pi
resulting from the PNPase in the Pi mop also prevented the detection of the small amount
(≤ 500 nM) of Pi produced in the single turnover reaction.
Suggestions for Overcoming Limitations of MDCC-PBP Pre-Steady-State Assay. This
technical limitation stemming from the Pi “mop” has been addressed by Martin Webb and
colleagues (106). They remedied the problem by using a potent PNPase inhibitor called
acyclovir diphosphate (Kd = 12 nM) (107). After weighing the cost/benefit analysis of
the synthesis of acyclovir diphosphate, we decided not to pursue the inhibitor as a
solution. Even if acyclovir diphosphate could remove PNPase activity in the Lon
ATPase reaction thus allowing for the detection of Pi release in the pre-steady-state an
actual rate constant for Pi release would not necessarily be determined. The MDCC-PBP
stopped flow experiment can only confirm that the Lon:Pi intermediate does not limit the
139
0.1
relative fluorescence
0
-0.1
-0.2
-0.3
-0.4
-0.5
0
0.2
0.4
0.6
0.8
1
time, (s)
Figure 5.11 – Detection of hydrolysis of 300 µM ATP by 5 µM Lon in the presence of
increasing amounts of Pi “mop” by fluorescent stopped flow (excitation 430 nm). (○)
0.05 unit/mL PNPase, 150 µM 7-MEG; (□) 1 unit/mL PNPase, 300 µM 7-MEG; (◊) 2
unit/mL PNPase, 600 µM 7-MEG; (x) 4 unit/mL PNPase, 1.2 mM 7-MEG.
140
reaction. This is determined by comparing the stopped flow and acid quench time
courses. If the two time courses are identical to one another, then the Lon:Pi does not
limit the reaction. The enzyme phosphodeoxyribose mutase can also be used to enhance
the efficiency of the Pi mop (106). Phosphadeoxyribose mutase (PDRM) catalyzes the
conversion of the product of the PNPase reaction, ribose-1-phosphate, to ribose-5phosphate in the presence of the co-factors Mn2+ and glucose-1,6,-bisphosphate. This
prevents the cycling of Pi from the slow hydrolysis of ribose-1-phosphate, and enhances
the ability of the Pi mop system to remove excess Pi from MDCC-PBP. Finally, Brune
and colleagues developed more stringent expression, purification, and labeling conditions
for MDCC-PBP which if implemented could increase the overall signal change in the
presence of Pi (96). If the use of MDCC-PBP were used to probe the pre-steady-state
activity of Lon in the future, one of these options would need to be employed to optimize
the system.
141
CHAPTER 6
THE FLUORESCENT NUCLEOTIDE ANALOGUE (MANT-ATP) IS USED TO
PROBE THE KINETIC MECHANISM OF ESCHERICHIA COLI LON
PROTEASE
142
Introduction
In the previous chapters pre-steady-state kinetic experiments were used to probe
the hydrolysis activities of the high- and low-affinity ATPase sites in E. coli Lon
protease. I established that the low-affinity ATPase sites display a pre-steady-state burst
in ADP production (~12 s-1) which occurs prior to initial peptide cleavage (77, 94)
(Chapter 3). Furthermore, the high-affinity sites hydrolyze ATP very slowly (~0.01 s-1)
and this hydrolysis is independent of nucleotide binding or hydrolysis at the low-affinity
sites (94) (Chapter 4). Other steps in the ATP hydrolysis pathway including binding, and
release of products have yet to be examined. Unlike the hydrolysis step that was probed
using a rapid chemical quench, which is a discontinuous experimental method of
obtaining kinetic data, a stopped flow instrument was used to visualize the binding and
release of nucleotide. The stopped flow, similar to the rapid quench, mixes the enzyme
and substrate within milliseconds allowing the reaction to be monitored on a time scale
from milliseconds to seconds. However, unlike the rapid quench, the stopped flow is
equipped to monitor changes in optical signal, store them, and graphically display the
time course at the end of the reaction (76). This continuous method for monitoring a
reaction relies on changes in fluorescence, light scattering, turbidity, or absorbance (63).
In order to monitor the binding and release of nucleotide from Lon in stopped
flow experiments we chose to utilize the fluorescent nucleotide analogue 2’-(or–3’)–O–
N-methylanthraniloyl (MANT-ATP). MANT-ATP is known to have an increase in
fluorescence quantum yield and a blue shift in its emission wavelength upon binding to
most proteins (108). MANT-ATP is a ribose-modified fluorescent analogue of ATP
(Figure 6.1). Although the structure in the figure represents the 2’-O isomer, both the 2’-
143
and 3’-O-(N-methylanthraniloyl) isomers exist in solution. Hiratsuka explains this is
because the 2’-hydroxyl group is kinetically more reactive for substitution whereas, the
esterification at the 3’-hydroxyl is more thermodynamicly favorable and 1,2-acyl shifts
interconvert the 2’-O- and 3’-O-isomers (108, 109). This chapter first establishes that
MANT-ATP is a suitable fluorescent analogue for the E. coli Lon protease system by
demonstrating that it is hydrolyzed and supports peptide cleavage comparably to ATP.
Then the kinetics of MANT-ATP binding and MANT-ADP release were measured using
pre-steady-state stopped flow experiments. The on rate of binding to the high- and lowaffinity ATPase sites was measured and found to be on the order of 105 M-1s-1. The rates
of MANT-ADP and MANT-AMPPNP binding were also monitored and were
comparable to MANT-ATP. A conformational change associated with nucleotide
binding was proposed in the stopped-flow experiments. This agreed well with the
conformational change detected in the tryptic digestion studies discussed in the previous
chapters. Two off rates associated with MANT-ADP release were detected and assigned
to the high- and low-affinity ATPase sites. The off rates from the high- and low-affinity
ATPase sites for MANT-ATP were also measured. Similar to the pre-steady-state burst
discussed in Chapter 3, no pre-steady-state rate constants determined using MANTnucleotides were dependent on S2 peptide or protein substrate. All of the pre-steady-state
rate constants determined were then incorporated into a kinetic model for ATPase activity
in E. coli Lon protease which was tested using computer simulation.
144
NH2
N
O
HO
P
O-
O
O
P
O-
N
O
O
P
N
O
N
O
OH
H
H
OH
O
C
H
O
NHCH 3
Figure 6.1 – Chemical Structure of MANT-ATP. Although this structure shows only the
2’-O isomer, in reality a mixture of both the 2’- and 3’-O-(N-methylanthraniloyl)
derivatives exist in solution.
145
Materials and Methods
Materials. Nucleotides were purchased from Sigma or ICN Biomedical. Tris, HEPES,
SBTI, and TPCK-treated trypsin were purchased from Fisher. MANT-AMPPNP was
purchased from Molecular Probes.
General Methods. Peptide synthesis and protein purification procedures were performed
as described previously (53). Synthesis of MANT-ATP and MANT-ADP was performed
by Xumei Zhang as described previously (108, 110). All enzyme concentrations were
reported as Lon monomer concentrations. All reagents are reported as final
concentrations. Unless otherwise stated all experiments were performed at 37 º C.
Steady-State MANT-ATPase Assays using Malachite Green. Steady-state velocity data
for MANT-ATP and ATP were collected using a modified colorimetric assay to detect
the release of inorganic phosphate (Pi) from the nucleotide tri-phosphates as the gamma
phosphate was cleaved by Lon protease (63, 64). Solutions containing 0.045% (w/v)
malachite green oxalate (MG) in deionized water, 4.2% (w/v) ammonium molybdate
(AM) in 4 N HCl, 2% Triton X-100 in deionized water, and 34% (w/v) sodium
citrate•2H2O in deionized water were prepared. Prior to each NTPase assay a 3:1 mixture
of MG:AM was made, stirred for at least 20 min, and filtered through 0.4 µM filter paper.
The Triton X-100 solution was then added to the MG/AM solution in the amount of 100
µL per 5 mL of 3:1 MG/AM solution. A solution of NaHPO4 and NaH2PO4 (pH 8.1) was
used as a calibration standard.
For the NTPase measurements, a 185 µL reaction mixture containing 50 mM Tris
buffer pH 8.1, 5 mM magnesium acetate (Mg(OAc)2), 2 mM DTT, various
concentrations of Lon protease (200 nM for intrinsic and peptide stimulated ATP and
146
peptide stimulated MANT-ATP, 1 µM for intrinsic MANT-ATP) as well as 500 µM
peptide substrate (S2, the nonfluorescent analogue of S3) for the peptide stimulated
NTPase assays was used. For all assays the NTPase reaction was initiated with the
addition of the nucleotide to the reaction mixture, and all reactions were performed at
least in triplicate. At 8 time points (from 0 min to 15 min) a 25 µL aliquot was
thoroughly mixed with 400 µL of MG/AM/Triton X solution. After 30 s, 50 µL of 34%
sodium citrate was added for color development. The absorbance was then recorded at
660 nm on a UV-vis spectrophotometer for each of the time points. The amount of Pi
formed at each time point was determined by comparing the absorbance of the sample to
a Pi calibration curve. Initial velocities were determined from plots of the amount of Pi
released versus time. The kinetic parameters were determined by fitting the averaged rate
data with eq 1 using the nonlinear regression program KaleidaGraph (Synergy) version
3.6.
k obs =
k cat , ATP [ ATP ]
K m + [ ATP ]
(1)
where kobs is the observed rate constant, kcat,ATP is the maximal rate constant, [Nu] is the
concentration of nucleotide, and Km is the Michaelis-Menten constant equal to the
concentration of ATP or MANT-ATP required to reach one half the maximal rate
constant.
Steady-State MANT-ATPase Assays using MDCC-PBP. Steady-state velocity data for
MANT-ATP and ATP were also measured using an assay to detect inorganic phosphate
(Pi) release as described previously (95, 102). Reactions contained 50 mM Tris at pH
8.0, 5 mM Mg(OAc)2, 2 mM DTT, 150 µM 7-methylguanosine (MEG), 0.05 unit/mL
147
PNPase, 300 nM or 150 nM E. coli Lon, + 500 µM S2 peptide, and 25 µM - 1 mM
MANT-ATP or ATP. ATPase activity was monitored on a Fluoromax 3
spectrofluorimeter (Horiba Group) where fluorescent MDCC-PBP was excited at 425 nm
and emitted at 465 nm. The velocity reactions were equilibrated at 37 °C for one minute
and initiated with the addition of Lon. Initial velocities were determined from plots of
the amount of Pi released versus time. All assays were performed at least in triplicate,
and the kinetic parameters were determined by fitting the averaged rate constant data
with equation 1 using the nonlinear regression program KaleidaGraph (Synergy) version
3.6.
Peptidase Methods. Peptidase activity was monitored on a Fluoromax 3
spectrofluorimeter (Horiba Group) as described previously (40). Assays contained 50
mM HEPES pH 8.0, 75 mM KOAc, 5 mM DTT, 5 mM Mg(OAc)2, 200 nM E. coli Lon,
150 µM MANT-ATP or ATP, 50-150 µM S1 peptide (100% fluorescent), and 200 µM 1.5 mM S3 peptide (10% fluorescent S1 peptide, 90% non-fluorescent analog S2 peptide)
(excitation 320 nm emission 420 nm). Initial velocities were determined from plots of
relative fluorescence versus time. All assays were performed at least in triplicate, and the
kinetic parameters were determined by fitting the averaged rate constant data with
equation 2 using the nonlinear regression program KaleidaGraph (Synergy) version 3.6.
k [S ]
k = max
n
K ' + [S ]
n
(2)
where k is the observed rate constant being measured in units of s, kmax is the maximum
rate constant referred to as kss,S3 in units of s-1, [S] is the variable peptide substrate in units
of µM, K' is the Michaelis constant for S, and n is the Hill coefficient. The Ks (µM) is
148
calculated from the relationship log K' = n log Ks, where Ks is the [S] required to obtain
50% of the maximal rate constant of the reaction referred to as Km,ATP or Km,MANT-ATP.
Tryptic Digestions. Tryptic digest reactions were monitored as described previously (55,
94). Briefly, 1.4 µM Lon in a reaction mixture containing 50 mM HEPES (pH 8.0), 5
mM Mg(OAc)2, 2 mM DTT, + 800 µM S2 peptide, and 1 mM ATP, 1 mM MANT-ATP,
1 mM ADP, 1 mM MANT-ADP, 1 mM AMPPNP, or 1 mM MANT-AMPPNP were
started by the addition of 1/50 (w/w) TPCK (N-p-tosyl-L-phenylalanine chloromethyl
ketone)-treated trypsin with respect to Lon. At 0 and 30 min, a 3 µL reaction aliquot was
quenched in 3 µg of soybean trypsin inhibitor (SBTI) followed by boiling. The quenched
reactions were resolved by 12.5% SDS-PAGE analysis and visualized with Coomassie
brilliant blue.
MANT-ATP Binding Time Courses by Fluorescent Stopped Flow. Pre-steady-state
experiments were performed on a KinTek Stopped Flow controlled by the data collection
software Stop Flow version 7.50 β. The sample syringes were maintained at 37ºC by a
circulating water bath. Syringe A contained 5 µM E. coli Lon monomer with and without
500 µM S2 peptide, 5 mM Mg(OAc)2, 50 mM HEPES pH 8, 75 mM KOAc, and 5 mM
DTT. Syringe B contained varying amounts of MANT-ATP, MANT-dATP, MANTADP, or MANT-AMPPNP (1-100 µM), 5 mM Mg(OAc)2, 50 mM HEPES pH 8, 75 mM
KOAc, and 5 mM DTT. MANT-nucleotide binding was detected by an increase in
fluorescence (excitation 360 nm emission 450 nm) resulting from rapid mixing of the
syringe contents in the sample cell. The resulting exponential data were a result of
averaging at least four traces. All experiments were performed at least in triplicate. The
averaged time courses were fit with equation 3
149
(
) (
Y = A1 exp − k1t + C1 + A2 exp − k 2 t + C2
)
(3)
where t is time in seconds, A1 and A2 are amplitudes for the first and second exponential
phases respectively in relative fluorescence units, k1 and k2 are the observed rate
constants for the first and second exponential phases in seconds, and C1, C2 are constants.
MANT-ADP Release Time Courses by Fluorescent Stopped Flow. Pre-steady-state
experiments were performed on a KinTek Stopped Flow controlled by the data collection
software Stop Flow version 7.50 β. The sample syringes were maintained at 37 º C by a
circulating water bath. Syringe A contained 5 µM E. coli Lon monomer with and without
500 µM S2 peptide which was pre-incubated with varying amounts of MANT-ADP (10
min), MANT-ATP, MANT-dATP (30 min, 37ºC) (0.05 - 200 µM), in 5 mM Mg(OAc)2,
50 mM HEPES pH 8, 75 mM KOAc, and 5 mM DTT. Syringe B contained 1 mM ADP,
5 mM Mg(OAc)2, 50 mM HEPES pH 8, 75 mM KOAc, and 5 mM DTT. MANT-ADP
release was detected by a decrease in fluorescence (excitation 360 nm emission 450 nm)
resulting from rapid mixing of syringe contents in the sample cell. The resulting
exponential data were a result of averaging at least four traces. All experiments were
performed in triplicate and the averaged time courses fit with equation 3.
Double mixing experiments were performed as above with the following
exceptions. The valve for syringe C was opened on the KinTek Stopped Flow, and the
delay line calibrated at 33 µL resulting in a second push volume of 46 µL. Syringe A
contained 5 mM E. coli Lon monomer and 5 mM ADP in 5 mM Mg(OAc)2, 50 mM
HEPES pH 8, 75 mM KOAc, and 5 mM DTT. Syringe B contained 1 mM ADP with or
without 500 µM S2 in 5 mM Mg(OAc)2, 50 mM HEPES pH 8, 75 mM KOAc, and 5 mM
DTT. Syringe C contained 100 µM MANT-ADP or ATP in 5 mM Mg(OAc)2, 50 mM
150
HEPES pH 8, 75 mM KOAc, and 5 mM DTT. The contents of Syringe A and C were
rapidly mixed with a first reaction time of 35 or 50 s for MANT-ATP and 3 ms for
MANT-ADP. The second push would then mix the developed reaction from syringes A
and C with the contents of Syringe B in the observation cell in order to monitor the
release of MANT-ADP from Lon over 30 s. All experiments were performed at least in
triplicate, and the averaged time courses fit with a single exponential equation.
151
Results and Discussion
MANT-ATP displays an increase in fluorescence when bound to Lon protease. Figure
6.2 shows that the fluorescent signal is approximately 1.5 times higher when 10 µM
MANT-ATP is bound to 2 µM Lon compared to 10 µM MANT-ATP in buffer. No
increase in fluorescence is detected unless enzyme is added to the reaction. As the Ki for
ADP has been reported at 300 nM (40) it binds to Lon more tightly than ATP. As shown
in Figure 6.2, when 10 µM ADP is added to the cuvette containing Lon bound to MANTATP, the fluorescent signal is abolished. This indicates that the fluorescent signal is a
result of a specific interaction between the nucleotide binding domain in Lon protease
and MANT-ATP.
Steady-State Assay for ATPase Activity Indicates MANT-ATP is a Reasonable ATP
Analog. In order to confirm that MANT-ATP was a substrate of Lon protease, the
steady-state kinetics of MANT-ATP hydrolysis were in examined and compared to those
of ATP. Both a discontinuous and a continuous assay were used to measure the steadystate rate constants of MANT-ATP hydrolysis. The discontinuous malachite green
colorimetric assay, described in detail in Chapter 2, monitors the release of inorganic
phosphate (Pi). The continuous fluorescent MDCC-PBP assay, described in detail in
Chapter 5, also monitors the release of inorganic phosphate (Pi). Figure 6.3 shows a plot
of the kobs as a measure of Pi production from the malachite green assay as a function of
nucleotide concentration. The data were fit with the Michaelis Menten equation and the
kinetic parameters are summarized in Table 6.1. According to this assay, the kcat,ATP and
Km,ATP values for MANT-ATP are lower than ATP however, the stimulation of the rate
152
relative fluorescence
8 10
5
7 10
5
6 10
5
5 10
5
4 10
5
3 10
5
2 10
5
1 10
5
0
380
400
420
440
460
480
500
520
wavelength (nm)
Figure 6.2 – Emission scans at excitation 360 nm of 10 µM MANT-ATP alone (○), 10
µM MANT-ATP bound to 2 µM Lon (□). The signal is specific to the binding
interaction because if enzyme storage buffer instead of Lon is added (◊) no increase in
fluorescence is detected. Furthermore 10 µM unlabeled ADP competes with MANTATP for binding to Lon and the increase in fluorescence is lost (X).
153
0.4
0.35
0.3
0.2
k
obs
-1
(s )
0.25
0.15
0.1
0.05
0
0
200
400
600
800
1000
1200
[Nucleotide], µM
Figure 6.3 – Steady-state MANT-ATPase and ATPase activity of Lon as determined
using the malachite green discontinuous colorimetric assay described in the Materials and
Methods. The concentrations of MANT-ATP and ATP that were used were 25, 50, 100,
250, 500 µM and 1 mM. The kinetic parameters (Table 6.1) were obtained by fitting the
data with the Michaelis-Menten equation. The kobs values for MANT-ATP hydrolysis
were determined in the absence (●) and presence of 800 µM S2 peptide (x). The kobs
values for ATP hydrolysis were also determined in the absence (■) and presence of 800
µM S2 peptide (♦) for comparison.
Table 6.1: Comparison of the Steady-State Parameters Associated with the Hydrolysis of
ATP and MANT-ATP
-1
kcat,ATP (s )
Km,ATP (µM)
*
ATP
0.10 + 0.01
MANT-ATP
0.02 + 0.01
0.37 + 0.01*
0.13 + 0.03*
48 + 10
4.3 + 9.1
52 + 15*
20 + 11*
values determined in the presence of S2 peptide
154
constants in the presence of S2 peptide is comparable. As discussed in Chapter 2, the
malachite green assay is not the optimal method for determining ATPase activity in the
Lon system because of both the high background Pi contamination and the error that is
inherent in a discontinuous assay. Therefore, the steady-state characterization of MANTATPase activity was determined using the MDCC-PBP coupled assay system on a
Fluoromax 3 spectrofluorimeter (Horiba Group) as described in Chapter 5. This assay
was developed by Martin Webb and colleagues (National Institute for Medical Research)
(95, 97, 102). MDCC-PBP binds Pi resulting in an increase in fluorescence thus allowing
for the rapid measurement of micromolar concentrations of Pi released from Lon in real
time. The observed rate constants were measured at varying concentrations of nucleotide
from the linear region of initial velocity plots of Pi released over time. When plotted
versus the concentration of nucleotide, both ATP and MANT-ATP yielded MichaelisMenten kinetics as shown in Figure 6.4. The kcat/Km profile was performed in the
absence and presence of S2 peptide for both ATP and MANT-ATP. The steady-state
kinetic parameters are summarized in Table 6.2. The MDCC-PBP assay was superior to
the malachite green assay in detecting Pi release because the kinetic parameters for
MANT-ATP were on the same order of magnitude with those obtained using the
sensitive discontinuous radioactive assay described in Chapter 2. Since the kcat/Km values
shown in Table 6.2 are similar for MANT-ATP and ATP, we concluded that Lon
hydrolyzes the fluorescent analog in a comparable manner to ATP.
Steady-State Assay Shows that MANT-ATP Supports Peptide Cleavage in Lon. In
addition to demonstrating that Lon hydrolyzes MANT-ATP comparably to ATP, it was
necessary to ensure that MANT-ATP was also capable of supporting S3 peptide
155
1.2
1
0.6
k
obs
-1
(s )
0.8
0.4
0.2
0
0
200
400
600
800
1000
[Nucleotide], µM
Figure 6.4 - Steady-state kinetics of ATP and MANT-ATP hydrolysis by E. coli Lon
protease. ATPase activity was monitored using a couple assay system where MDCCPBP binds the Pi released from the hydrolysis of ATP and MANT-ATP by Lon resulting
in an increase in fluorescence over time. The concentrations of MANT-ATP and ATP
that were used were 25, 50, 100, 250, 500 µM and 1 mM. The kinetic parameters (Table
6.2) were obtained by fitting the data with the Michaelis-Menten equation. The kobs
values for MANT-ATP hydrolysis were determined in the absence (●) and presence of
500 µM S2 peptide (■). The kobs values for ATP hydrolysis were also determined in the
absence (♦) and presence of 500 µM S2 peptide (▲) for comparison.
Table 6.2: MANT-ATP and ATP Steady-State Kinetic Parameters Associated with ATP
Hydrolysis
Intrinsic
ATPase
Intrinsic
MANT-ATPase
S2 Stimulated
ATPase
S2 Stimulated
MANT-ATPase
kcat (s-1)
0.34 + 0.01
0.53 + 0.02
1.1 + 0.1
1.1 + 0.1
Km (µM)
18 + 4
43 + 5
31 + 8
37 + 6
kcat/Km (103 M-1s-1)
19
12
35
30
156
cleavage. Lee and Berdis developed a continuous fluorescent peptidase assay to monitor
the kinetics of peptide cleavage as described in the introduction (53). Because of the
inner-filter effect of fluorescence there is interference at high concentrations of 100%
fluorescent S1 peptide. Therefore we use S3 peptide, a 10% mixture of fluorescently
labeled peptide with its nonfluorescent analogue, S2. This 10 amino acid long (S3 and
S2) peptide sequence contains only one cleavage site and comes from the λN protein,
which is a physiological substrate of E. coli Lon. Because our model peptide substrate
contains only one Lon cleavage site, is unstructured, and stimulates ATP hydrolysis, its
kinetics of degradation can be directly attributed to the ATP-dependent peptidase reaction
rather than polypeptide unfolding or processive peptide cleavage (40, 53). The observed
steady-state rate constants of S3 cleavage (kss,S3) were determined at varying
concentrations of S3 (50 µM – 1.5 mM) and 150 µM MANT-ATP or ATP. Under these
conditions, the predominant enzyme form is Lon-ATP, so the observed rate constants are
a reflection of the effect of nucleotide hydrolysis rather than binding. The S3 hydrolysis
reactions were monitored by the increase of fluorescence over time, which when
calibrated reflects the amount of peptide hydrolyzed over time. The observed steadystate rate constants were then plotted as a function of S3 concentration and this yielded a
sigmoidal plot as shown in Figure 6.5. The kinetic parameters obtained from fitting the
data in Figure 6.5 with the Hill equation are summarized in Table 6.3. Although the
value of n determined here is slightly higher than that determined previously (n=1.6) (40,
55) both are approximately equal to 2. The discrepancy is most likely due to slightly
differing experimental methods. Unlike the previous assays which were performed with
S3 at all concentrations of peptide, the excitation of the MANT fluorophore was
157
10.0
kobs (s-1)
7.5
5.0
2.5
0.0
0
250
500
750 1000 1250 1500 1750
[S3], µM
Figure 6.5 – 150 µM ATP and MANT-ATP dependent peptidase activity was monitored
using a modified FRET assay system where the cleavage of the S3 peptide containing a
fluorescent donor and quencher results in donor/quencher separation and an increase in
fluorescence over time. The initial steady-state rates of S3 cleavage were obtained from
the time courses of peptide cleavage at varying [S3], and converted to steady-state rate
constants kss,S3 by dividing by the [Lon]. Both ATP- (●) and MANT-ATP (■) –mediated
S3 cleavage yielded sigmoidal curves which were fit with the Hill equation. The
resulting kinetic parameters are summarized in Table 6.3.
Table 6.3: MANT-ATP and ATP Steady-State Kinetic Parameters Associated with S3
Cleavage
kss,S3
(s-1)
Km
(µM)
n
ATP
9.6 + 0.2
240 + 9
2.4 + 0.2
MANT-ATP
5.9 + 0.3
360 + 30
2.5 + 0.4
158
interfering with peptide fluorescent signal at lower concentrations of S3, so it was
necessary for the 100% fluorescent analog S1 to be used for the peptide concentrations
from 50 – 150 µM. The peptidase assay was performed at a single concentration of
MANT-ATP and ATP for the purpose of comparison. As MANT-ATP was found to
support peptide cleavage similar to ATP no further concentrations were examined in
order to conserve the fluorescent analog.
Tryptic Digestion Reactions Show that MANT-ATP Supports Nucleotide Dependent
Conformational Change in Lon. As discussed in Chapter 2 we utilized limited tryptic
digestion to probe the functional role of nucleotide binding to Lon. This revealed an
adenine-specific conformational change associated with nucleotide binding, which can
primarily be monitored by the stability of a 67 kDa fragment of Lon. When sequenced,
this fragment was composed of the ATPase, SSD, and protease domains of Lon. To
ensure that the introduction of the fluorophore on MANT-ATP does not affect the
nucleotide’s ability to induce this conformational change in Lon, 1.4 µM Lon was
subjected to limited tryptic digestion (1/50, w/w) in the presence of no nucleotide and
saturating amounts (1 mM) of ATP, MANT-ATP, ADP, MANT-ADP, AMPPNP, and
MANT-AMPPNP. At 0 and 30 minutes the reaction was quenched with soybean trypsin
inhibitor (SBTI) and resolved with 12.5% SDS-PAGE as shown in Figure 6.6. This
figure demonstrates that MANT-ATP (lanes 4-5) induces the same conformational
change as ATP (lanes 2-3). MANT-ADP (lanes 10-11) and MANT-AMPPNP (lanes 1415) also induce the same conformational change as ADP (lanes 8-9) and AMPPNP (lanes
12-13) respectively. S2 peptide did not change the digestion pattern (gel not shown)
159
No Nu
0 30
ATP
MANT
MANT
MANT
ATP ADP ADP AMPPNP AMPPNP
0 30 0 30 0 30 0 30 0 30 0 30
Intact Lon
67 KDa fragment
SBTI
Figure 6.6 – SDS-PAGE visualized by Coomassie brilliant blue shows 1.4 µM Lon
digested with a limiting amount of trypsin and quenched with SBTI at the indicated times
as described in Materials and Methods. Lane 1 shows the molecular markers in
kilodaltons (from top to bottom): 183,114, 81, 64, 50, 37, 26, 20. Lanes 2-3 contain Lon
without nucleotide, lanes 4-5 contain Lon + 1 mM ATP, lanes 6-7 contain Lon + 1 mM
MANT-ATP, lanes 8-9 contain Lon + 1 mM ADP, lanes 10-11 contain Lon + 1 mM
MANT-ADP, lanes 12-13 contain Lon + 1 mM AMPPNP, and lanes 14-15 contain Lon +
1 mM MANT-AMPPNP.
160
indicating that peptide does not induce any conformational change that is detectable by
tryptic digestion.
MANT-ATP Used to Probe Binding Interactions with Lon. MANT-ATP was also used to
probe the Kd values of the high- and low-affinity ATPase sites on a Fluoromax 3
spectrofluorimeter (Horiba Group). Many experimental conditions were attempted to
optimize the signal to noise ratio. In these experiments the concentration of MANT-ATP
was varied from 1 to 100 µM. The concentration of enzyme in Kd titrations should be
kept at or below the Kd level of 10 µM (38, 72). Although the concentration of enzyme
was varied from 125 nM to 2 µM, the background signal from the increasing amounts of
MANT-ATP proved to be an unavoidable problem. If the concentration of Lon was
increased to 10 µM, a sufficient change in signal was obtained. This binding isotherm is
shown in Figure 6.7, and resulted in a Kd of 9 + 1 µM. However, at this concentration of
enzyme it is ambiguous whether a true Kd is being detected. It is more likely that the
amount of Lon present in the reaction is being titrated. Furthermore because Lon is
incubated with MANT-ATP in these experiments, the resulting binding isotherm is more
likely a reflection of MANT-ADP binding which should have a Kd value closer to the
published value of 300 nM (38, 40).
Optimizing Protein Purification Protocol to Increase the Yield. The MANT-nucleotides
were also utilized in pre-steady-state stopped flow experiments to probe individual steps
including nucleotide binding and release. In order to perform the many pre-steady-state
experiments, large quantities of E. coli Lon were needed because the enzyme
concentration and reaction volume in pre-steady-state experiments is higher than in
steady-state experiments. Therefore, the expression and purification of E. coli Lon was
161
6
1.4 10
6
relative fluorescence
1.2 10
1 10
6
8 10
5
6 10
5
4 10
5
2 10
5
0
0
10
20
30
40
50
[MANT-ATP], µM
Figure 6.7 – Determining the Kd for the low-affinity ATPase site in E. coli Lon using
MANT-ATP. 10 µM Lon was incubated with varying amounts of MANT-ATP from 0 –
100 µM . The generated data were fit using a binding isotherm equation 1, and the
resulting Kd value was 9 + 1 µM.
162
modified to increase the yield by approximately five fold. This was accomplished by
using a pET vector for expression which is driven by a T7 promoter. The amount of ion
exchange chromatography resin used to purify Lon was also optimized so that for every
4.5 L of culture grown, 50 g of cation and anion exchange resin is used. These
modifications increased the yield of E. coli Lon to ~ 11 mg of Lon per liter of culture.
Monitoring the Rate of MANT-ATP Binding to Lon. Using the fluorescent nucleotide
analogue MANT-ATP and the increased amounts of Lon, pre-steady-state kinetic
experiments were performed on the stopped flow to study individual steps along the Lon
reaction pathway. The on rate of MANT-ATP binding was monitored by rapidly mixing
5 µM Lon with amounts of MANT-ATP varying from 1 µM to 100 µM. These
experiments were performed in the absence and presence of 500 µM S2 peptide although
the presence of peptide had no effect on MANT-ATP binding. A representative time
course for MANT-ATP binding is shown in Figure 6.8a and all time courses were best fit
using a double exponential equation. Two observed rate constants (kon,1, kon,2) are
extracted from the double exponential equation and plotted versus the concentration of
MANT-ATP as shown in Figure 6.8b. The rate constants associated with MANT-ATP
binding are identical in the presence and absence of S2 peptide as shown in Figure 6.8b
indicating that peptide has influence on the binding interaction of nucleotide with Lon.
The observed rate constants, kon,1, are linearly dependent on the concentration of MANTATP (Figure 6.8b). The second order rate constant associated with MANT-ATP binding
(6.8 x 105 M-1s-1) is extracted from the slope of the line in Figure 6.8b and the values are
summarized in Table 6.4. An estimate of the off rate (11 s-1) can be determined from the
y-intercept (Table 6.4). When a Kd is calculated by dividing the off rate by the on rate,
163
1.2
a
relative fluorescence
1
0.8
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
time, (s)
b
80
k
obs
-1
(s )
60
40
20
0
0
20
40
60
80
100
[MANT-ATP], µM
Figure 6.8 – (a) Representative time course of MANT-ATP binding to Lon. Five
micromolar Lon was rapidly mixed with varying amounts of MANT-ATP (excitation 360
nm, emission 450 nm) in the presence and absence of 500 µM S2 peptide and the
increase in fluorescence was monitored for 1 second. The time courses were fit with a
double exponential equation (eq. 3, solid black line). The two resulting rate constants
(kon,1, kon,2) are shown in (b) at varying concentrations of MANT-ATP. (b) On-rate
constants of MANT-ATP binding plotted versus the concentration of MANT-ATP. kon,1
shows a linear dependence on [MANT-ATP] both in the absence (●) and presence (□) of
500 µM S2 peptide. The second order rate constant associated with MANT-ATP binding
can be estimated from the slope of the line (kon,MANT-ATP = 6.8 x 105 M-1s-1). The second
rate for MANT-ATP remains constant at 5 s-1, and likely indicates a conformational
change associated with nucleotide binding. The rate constants are summarized in Table
6.4.
164
the value (16 µM) is similar to the previously published value of 10 µM for the lowaffinity ATPase site (38, 94). The range of nucleotide concentration needed to probe the
high-affinity site (0.05 – 5 µM) was beyond the limit of detection at the lower
concentrations and indistinguishable from the low-affinity site at the higher
concentrations. The observed rate constant kon,2 ~ 5 s-1 (Figure 6.8b, Table 6.4) shows no
dependence on the concentration of MANT-ATP. The lack of dependence on nucleotide
concentration could be indicative of a unimolecular event such as a conformation change
associated with nucleotide binding, which is in agreement with the tryptic digestion study
shown in Figure 6.6. Identical stopped flow binding experiments were performed with
both MANT-ADP and MANT-AMPPNP and the results are summarized in Table 6.4.
All of the adenine nucleotides bound to Lon at comparable rates on the order of (105 M1 -1
s ). Although the rate of nucleotide binding is fast compared to the other rate constants
measured in the Lon pathway, it is significantly slower than diffusion control (108 M-1s1
). The reduction in the rate of nucleotide binding could either be due to the presence of
glycerol in the reaction (111), or that the rate of binding is coupled with the slow (5 s-1)
conformational change which is needed to detect an increase in fluorescence from
MANT-ATP.
During the synthesis of MANT-ATP, the MANT fluorophore reacts with both the
2’ and 3’ hydroxyl on the ribose (108). The 2’and 3’ isomers of the MANT-nucleotides
are not separated and therefore both exist in solution (108). It has also been shown that
the esterification of the fluorophore can cause changes in fluorescence (112). Because
the 2’-deoxynucleotides have an available hydroxyl group only at the 3’ position this
complication does not arise when dATP is labeled with MANT. Therefore, control
165
Table 6.4: Rate Constants Associated with Adenine Nucleotide Binding as Determined
by Fluorescent Stopped Flow
MANT-Nucleotide
kon,MANT-Nu
(105 M-1s-1)
koff, MANT-Nu
(s-1)
kon,2
(s-1)
ATP
6.8
11
4.1 + 1.2
ATP + S2
6.8
10
3.7 + 1.2
ADP
6.9
3.9 + 0.9
ADP + S2
6.1
AMPPNP
3.2
AMPPNP + S2
2.1
9.5
0.46 (0.13)*
9.7
0.44* (0.13)*
22
6.6* (0.16)*
24
6.7* (0.16)*
*
*
3.7 + 0.9
6.7 + 2.8
7.1 + 4.7
Values determined by single mixing release rate experiments where Lon was pre-incubated with MANT
nucleotide and then rapidly mixed with excess unlabeled nucleotide. These values are more accurate
because the off rates are being directly measured in the experiment. Two off rates for the nucleotides are
detected in this experiment. The off rate from the high-affinity site is in parenthesis and the other number
represents the off rate from the low-affinity site.
166
1.2
relative fluorescence
1
0.8
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
time, (s)
Figure 6.9 – Representative time course of 50 µM MANT-dATP binding to 5 µM Lon.
The time course is double exponential in nature, ensuring that the esterification of the
flourophore is not causing a detectable change in fluorescence.
Table 6.5: Comparison of the Rate Constants Associated with MANT-ATP Binding and
MANT-ADP Release with MANT-dATP
MANT-ATP
MANT-dATP
MANT-ADP
MANT-dADP
kobs, 1 (s-1)
27 + 2
26 + 2
0.45 + 0.01
0.37 + 0.02
kobs, 2 (s-1)
4 + 0.5
5+1
0.12 + 0.01
0.12 + 0.01
167
experiments were performed for binding of MANT-dATP to ensure that the biphasic time
courses were not a result of an esterification of the fluorophore on the MANTribonucleotides causing a change in fluourescence. A representative time course for the
binding of 50 µM MANT-dATP to 5 µM Lon with 500 µM S2 peptide is shown in
Figure 6.9. As with MANT-ATP, the time courses were biphasic in both the presence
and absence (data not shown) of 500 µM S2 peptide and the observed rate constants
summarized in Table 6.5.
Monitoring the Rate of MANT-ADP Release from Lon. The rate of MANT-ADP release
from Lon was also monitored using pre-steady-state stopped flow experiments. These
experiments were performed in a number of different ways to obtain as much information
as possible. First, various concentrations of MANT-ADP (500 nM – 200 µM) were preincubated with 5 µM Lon in both the absence and presence of 500 µM S2 peptide and
rapidly mixed with 1 mM unlabeled ADP. These experiments resulted in time courses
showing a decrease in fluorescence and were best fit using a double exponential equation.
A representative time course is shown in Figure 6.10. The two rate constants (koff,1, koff,2)
associated with MANT-ADP release are summarized in Table 6.6. As with all presteady-state rate constants measured thus far, S2 peptide has no effect on the rate
constants associated with MANT-ADP release. Furthermore, as one would expect for a
unimolecular reaction, the rate constants show no dependence of the concentration of
MANT-ADP. This experiment was also performed with MANT-AMPPNP to determine
an estimate of the rate of ATP release from the high- and low-affinity ATPase sites. The
off rate values for MANT-AMPPNP are summarized in Table 6.4. When these observed
off rates were divided by the on rate, the two Kd values obtained for AMPPNP were 0.3
168
0
relative fluorescence
-0.5
-1
-1.5
-2
-2.5
0
5
10
15
20
25
30
time (s)
Figure 6.10 – Representative time course of MANT-ADP release in single mixing
stopped flow experiments. Five micromolar Lon was pre-incubated with varying
amounts of MANT-ADP or MANT-ATP (excitation 360 nm, emission 450 nm) in the
presence and absence of 500 µM S2 peptide and rapidly mixed with 1 mM ADP. The
resulting decrease in fluorescence was monitored for 30 seconds, and the time courses
were fit with a double exponential equation (eq 3). Two average rate constants resulted
from the fit koff,1 = 0.46 + 0.03 s-1 and koff,2 = 0.14 + 0.01 s-1 and are summarized in Table
6.6.
169
µM and 9 µM. These values are in close agreement with the Kd values determined
previously (0.5 µM (94), 10 µM (38)). Therefore it is likely that the koff values of ATP
are the same as AMPPNP.
The experiment was also performed where saturating
amounts (15 µM) of ADP was pre-incubated with 5 µM Lon and rapidly mixed with 500
µM MANT-ADP. These experiments resulted in time courses displaying an increase in
fluorescence which is limited by the release of ADP. The two rate constants associated
with ADP release (koff,1, koff,2) are comparable to those obtained in the experiment
described above (data not shown). Thus the fluorescent label on the nucleotide does not
affect the off rate. Furthermore, these experiments were performed using MANT-ATP
which was pre- incubated with Lon in the absence and presence of S2 peptide for twenty
minutes to allow for hydrolysis. The off rate of the enzymatically generated MANTADP from Lon was then determined on the stopped flow. By performing the experiment
in this way, I was attempting to isolate the “post”-catalytic enzyme form, “F,” which was
proposed previously and described in detail in the introduction chapter (Scheme 1.2).
Identical off rates were obtained when the experiment was performed this way (Table
6.6) suggesting the “pre” and “post” catalytic forms of Lon release ADP identically.
However, the drawback of this experiment is that there is no substantial evidence that the
“F” form would be stable and not converted back to free enzyme following MANT-ATP
hydrolysis. The hypothesis that the “pre”- (“E”) and “post”- catalytic forms of Lon
release ADP differently will be described in Chapter 7. To ensure that the two
exponential phases in the time course were not a result of esterification of the MANT
fluorophore causing a change in fluorescence, control experiments were performed with
2’-deoxyMANT-ADP. As shown in Figure 6.11, the time courses in the MANT-dADP
170
6.8
6.6
relative fluorescence
6.4
6.2
6
5.8
5.6
5.4
5.2
0
5
10
15
20
25
30
time (s)
Figure 6.11 – Representative time course of MANT-dADP release in single mixing
stopped flow experiments performed identically to Figure 6.12. The time course is fit
with a double exponential equation (solid black line) demonstrating that the biphasic
nature of the time course is not a result of esterification of the fluorophore.
Table 6.6: Summary of MANT-ADP Release Rate Constants from Single Mixing
Stopped Flow Experiments
Intrinsic koff,1 (s-1)
S2 Stimulated koff,1 (s-1)
Intrinsic koff,2 (s-1)
S2 Stimulated koff,2 (s-1)
MANT-ADP MANT-ATP
MANT-ADP MANT-ATP
MANT-ADP MANT-ATP
MANT-ADP MANT-ATP
0.05
0.42 + 0.01
0.43 + 0.01
0.45 + 0.01
0.44 + 0.01
0.12 + 0.01
0.12 + 0.01
0.12 + 0.01 0.12 + 0.01
0.1
0.52 + 0.01
0.44 + 0.01
0.45 + 0.01
0.46 + 0.02
0.13 + 0.01
0.13 + 0.02
0.13 + 0.01
0.14 + 0.02
0.5
0.47 + 0.02
0.51 + 0.16
0.47 + 0.02
0.46 + 0.02
0.15 + 0.01
0.13 + 0.02
0.15 + 0.01
0.14 + 0.02
50
0.42 + 0.01
0.46 + 0.04
0.45 + 0.01
0.49 + 0.05
0.12 + 0.01
0.12 + 0.04
0.12 + 0.01
0.13 + 0.04
[MANTNu]
(µM)
200
0.46 + 0.06
a
N.D.
0.40 + 0.04
N.D.
N.D. : these values were not determined
171
0.14 + 0.01
N.D.
0.14 + 0.01
N.D.
experiments also fit with a double exponential equation thus ensuring that the biphasic
nature of the MANT-ADP release time courses was not a result of esterification of the
fluorophore. A comparison of the rate constants associated with MANT-dADP and
MANT-ADP release is shown in Table 6.5.
Isolating MANT-ADP Release from the Low-Affinity ATPase Site. Since the two rate
constants associated with MANT-ADP release are not a result of a fluorescent artifact
from the esterification of the fluorophore, experiments were performed in order to
determine what the two off rate constants were describing. Because two affinities (Kd’s)
(Chapter 4) for ATP exist and no difference in the on rate is observed, it is conceivable
that the two observed off rates were a result of the high- and low-affinity ATPase sites
releasing MANT-ADP at different rates. In order to probe the high-affinity ATPase site
low concentrations of MANT-ADP (50 nM – 500 nM) were used. Lon and MANT-ADP
were pre-incubated prior to mixing allowing for equilibrium to be established, and the
experiment is designed such that excess unlabeled ADP chases off all available MANTADP. Therefore, due to the sensitivity of the stopped flow the release of ADP from both
sites was being detected even at low concentrations of MANT-ADP (Table 6.6). In order
to uncouple low-affinity site MANT-ADP release from high-affinity MANT-ADP release
double mixing experiments were performed. In these experiments a third syringe is
opened allowing for the mixing of two reaction components for a designated period of
time prior to the introduction of the third component and subsequent monitoring of the
fluorescent signal. In order to block to high-affinity sites I again utilized the technique of
adding stoichiometric amounts of unlabeled ADP to Lon (Chapter 4) (94). Whether the
ADP was generated in situ or directly added in the pre-incubation with Lon, the pre-
172
steady-state ATPase activity at the low affinity sites remained unaffected. Therefore, this
technique was used to isolate MANT-ADP release from the low-affinity sites. All the
nucleotides bind to Lon very fast (~ 105 M-1s-1), and this is used to select for the lowaffinity ATPase sites in double mixing experiments. Lon:ADP is rapidly mixed (3 ms)
with MANT-ADP and the fluorescent ADP is subsequently chased off with 1 mM ADP.
Figure 6.12 shows the time course generated from this double mixing experiment.
Unlike the previous experiments which all resulted in double exponential time courses,
when the high-affinity sites were blocked with unlabeled ADP, a single exponential time
course is detected. The observed rate constant (koff,1 = 0.5 + 0.1 s-1) obtained from the fit
of the data with a single exponential equation is presumably describing the rate of
MANT-ADP release from the low-affinity ATPase sites. By inference the second rate
constant detected in the single mixing stopped flow experiments (koff, 2 = 0.14 s-1, Table
6.6) must describe the release of ADP from the high-affinity ATPase site. The presence
of S2 peptide had no effect on this observed rate constant. The double mixing
experiment was also performed using MANT-ATP. For these experiments the delay time
was increased first to 35 s and then to 50 s to allow for the complete hydrolysis of 150
µM MANT-ATP prior to chasing of MANT-ADP. Figures 6.13 a and b show
representative time courses for the 35 s and 50 s delay respectively. When the 35 s delay
time was used, two observed rate constants were observed (kobs,1 = 8 + 1 s-1, kobs,2 = 0.64 +
0.01 s-1). It is not clear from the experimental design whether the faster rate constant
describes MANT-ATP release or a stimulated MANT-ADP release. As kobs,2 is similar to
all of the rate constants describing MANT-ADP release from the low-affinity site it is
likely the same in this case. Because the results are ambiguous in this experiment, the
173
0
relative fluorescence
-0.05
-0.1
-0.15
-0.2
0
5
10
15
20
25
30
time (s)
Figure 6.12 – Representative time course of MANT-ADP release in double mixing
experiments. The high-affinity ATPase sites in 5 µM Lon were blocked with 5 µM ADP
and this mixture was rapidly mixed with 100 µM MANT-ADP for 0.003 s. 1 mM ADP
then mixes with the reaction to displace the bound nucleotide and the decrease n
fluorescence is monitored for 30 seconds. The time courses were fit with a single
exponential equation (solid black line), and the resulting rate constant (koff = 0.5 + 0.1 s-1)
describes the release of ADP from only the low-affinity sites.
174
a
0
relative fluorescence
-0.1
-0.2
-0.3
-0.4
-0.5
0
5
10
15
20
25
30
20
25
30
time (s)
0
b
relative fluorescence
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
0
5
10
15
time (s)
Figure 6.13 – Representative time courses of MANT-ADP release in double mixing
experiments. The high-affinity ATPase sites in 5 µM Lon were blocked with 5 µM ADP
and this mixture was rapidly mixed with 100 µM MANT-ATP for 35 s (a) or 50 s (b)
respectively. 1 mM ADP then mixes with the reaction to displace the bound nucleotide
and the decrease n fluorescence is monitored for 30 seconds. The time course in (a) was
fit with a double exponential equation (solid black line) resulting in two observed rate
constants (kobs,1 = 8 + 1 s-1, kobs,2 = 0.64 + 0.01 s-1). The time course in (b) was fit with a
single exponential equation (solid black line), and the resulting rate constant (koff = 0.5 +
0.1 s-1) describes the release of ADP from only the low-affinity sites.
175
delay time was increased to 50 s to ensure that all MANT-ATP present was hydrolyzed.
In this case a single exponential decay was detected described by an observed rate
constant of 0.33 + 0.01 s-1 which is presumably the rate constant for release of MANTADP from the low-affinity sites subsequent to MANT-ATP hydrolysis at the low-affinity
sites.
In summary MANT-labeled nucleotides were used as fluorescent probes to
determine binding affinities for Lon, as well as to compare the kinetics of ATP,
AMPPNP, and ADP binding to and release from E. coli Lon protease. The pre-steadystate binding experiments performed using the MANT-nucleotides verified that the on
rate of ATP binding is comparable to AMPPNP, and that a conformational change
accompanies nucleotide binding. Therefore, the difference between ATP and AMPPNPactivated peptide cleavage occurs after nucleotide binding. Two binding affinities for
ATP have been detected (38, 94). The binding experiments demonstrated that ATP binds
to the high- and low-affinity ATPase sites similarly (6 x 105 M-1s-1). However, the
MANT-AMPPNP release studies confirmed our expectation of two Kd values when two
off rates were detected (koff,1 = 6.6 s-1, koff,2 = 0.16 s-1). Two observed rate constants (koff,1
= 0.46 s-1, koff,2 = 0.14 s-1) were also detected in association with MANT-ADP release.
However, only one on rate was detected using MANT-ADP and only one affinity for
ADP has been reported. Most likely two Kd values were not detected for ADP because of
the modest (three fold) difference between the two observed off rates.
The steady-state kcat for ATP hydrolysis in Lon is stimulated in by peptide or
protein substrate (1, 39). However, a detailed explanation of how the peptide or protein
substrate interaction is stimulating ATPase activity is not available. Prior steady-state
176
studies suggested that ADP release is facilitated by the presence of peptide or protein
substrate (39, 40). Therefore, it was surprising that the rate constant for MANT-ADP
release was not increased in when peptide or protein was added to the reaction. In fact no
pre-steady-state rate constant associated with ATPase activity measured thus far is
affected by peptide or protein substrate. This will be discussed in more detail in Chapter
7. This information along with the compilation of the individual rate constants measured
in Chapters 3-6 led to the construction of a revised kinetic model for Lon protease. The
revised kinetic model for E. coli Lon protease is discussed in detail in Chapter 7.
177
CHAPTER 7
CONCLUSIONS AND FUTURE DIRECTIONS:
A REVISED KINETIC MODEL FOR THE ATP HYDROLYSIS ACTIVITY IN
ESCHERICHIA COLI LON PROTEASE
178
Introduction
In Chapters 2-6, I described a combination of steady-state and pre-steady-state
techniques utilized to test the validity of a proposed kinetic mechanism for Lon protease
shown in Scheme 1.2. Overall the studies provided unique mechanistic insight into how
Lon protease functions and are the basis for revised kinetic models for both intrinsic and
peptide stimulated ATPase activities. A revised kinetic model for intrinsic ATPase
activity in Lon is illustrated in Scheme 7.1. Although Lon exists as a higher order
oligomer which is most likely a hexamer (32, 113), it is represented as a dimer in order to
simplify the schemes. Each Lon monomer contains four defined domains, the amino
terminus, ATPase, substrate sensor and discriminatory (SSD), and the protease domain.
The amino terminus is not represented in the schemes for simplicity, the low-affinity
ATPase domains are represented as blue ovals, the high-affinity ATPase domains are
represented as red ovals, the SSD domain is represented as white ovals, and the peptidase
domain is represented as white circles. In Scheme 7.1, Lon with no substrate bound
(form I) binds to ATP at both the high- and low-affinity ATPase sites at a fast rate (Table
6.4, kon,ATP = 6.8 x 105 M-1s-1). The binding of nucleotide induces a conformational
change (form II) measured in the stopped-flow binding experiments at approximately 5 s1
(Table 6.4). ATP is subsequently hydrolyzed at the low-affinity ATPase sites at 11 s-1
(Chapter 3) (form IV), and ADP is released from the low-affinity ATPase sites at 0.5 s-1
(Table 6.6). Turnover at the low-affinity ATPase sites then occurs in the absence of
peptide (forms II-IV) at kcat = 0.5 s-1 (Table 6.2) and is limited by ADP release. In a
separate pathway (forms IV and V) the high-affinity ATPase sites can hydrolyze ATP at
0.01 s-1 (Chapter 4) and release ADP at 0.1 s-1 (Table 6.6).
179
koff,1 = 0.5 s-1
koff,2 = 0.1 s-1
ADP
central
cavity
kon,ATP = 6.8 x 105 M-1 s-1
I
koff,2 = 0.1 s-1
ADP
ADP
V
ATP
ATP
ADP
ATP
kcat = 0.5 s-1
ATP
ADP
kon,2 = 5 s-1
II
koff,1 = 0.5 s-1
kobs = 0.01 s -1
ATP
ADP
ATP
ATP
kburst = 11 s-1
IV
III
Form (I) represents oligomeric free enzyme shown for simplicity as a dimer (bottom white
circle = peptidase domain, middle white oval = substrate senor and discriminatory
domain (SSD), top left blue oval = low-affinity ATPase, top right red oval = high-affinity
ATPase)
Forms (II – IV) represent intrinsic steady state ATPase activity at the low-affinity sites
Form (V) respresents the ability of the high-affinity site to hydrolyze ATP
Scheme 7.1 – Revised catalytic mechanism for intrinsic ATPase activity.
180
The rapid quench studies described in Chapter 3 confirmed that ATP hydrolysis
occurs at the low-affinity ATPase sites prior to peptide cleavage (77, 94). Because burst
kinetics was detected in the pre-steady-state time course of ATP hydrolysis, the rate
limiting step must occur after ATP hydrolysis, and ADP release would be consistent with
this. The rate-limiting step should also have a rate constant similar to the overall kcat for
ATP hydrolysis (kcat, S2 stimulated = 1.1 s-1, kcat, intrinsic = 0.53 s-1). Furthermore when the
timing of all of the pre-steady-state experiments was examined in entirety, the highaffinity sites seem to be essentially catalytically inactive. The pulse-chase experiments
(Chapter 3 (77)) and single turnover experiments (Chapter 4 (94)) demonstrated that the
high-affinity ATPase sites are capable of hydrolyzing ATP if given enough time;
however, the steady-state rate is not affected by high-affinity site ATPase activity.
Although Lon (5 µM) undergoes multiple rounds of peptide cleavage at limiting (500
nM) ATP, 100 µM peptide is cleaved prior to the half-life of the high-affinity ATPase
site reaction (94). For these reasons it appears that the ATPase pre-steady-state activity
can be attributed to only the low-affinity sites. However, a detailed understanding of how
the S2 peptide interacts with Lon to stimulate steady-state ATPase activity is still
necessary. The steady-state kcat value can thus be attributed to the low-affinity site
ATPase reaction which is stimulated in the presence of peptide. Because the off rate of
MANT-ADP from the low-affinity site (0.46 s-1, Table 6.6 ) approaches the turnover
number for intrinsic MANT-ATPase activity (0.53 s-1, Table 6.2), ADP release is likely
the rate-limiting step in this pathway. The best estimate of a turnover number for the
high-affinity sites is 0.01 s-1 as determined in Chapter 4 (94). However, the high-affinity
ATPase sites release ADP at 0.14 s-1, which is faster than the turnover number. Thus
181
another step must be limiting turnover at the high-affinity sites. Until the activities at the
two sites can be uncoupled this step will likely remain unknown. Because hydrolysis is
so slow, it would not be surprising if the chemical reaction was rate-limiting for the highaffinity sites.
The proposed mechanism outlined in Scheme 7.1 only addresses ATP hydrolysis
in the absence of peptide or protein substrate. Although the presence of peptide has no
effect on the pre-steady-state rate constants measured thus far, it is known to stimulate
steady-state ATPase turnover (Chapter 2). Precisely how peptide or protein substrate is
accelerating the ATPase turnover is still unknown. Prior experiments were performed
which suggested that the presence of peptide of protein substrate facilitates the release of
ADP from Lon. Menon and Goldberg demonstrated this in two ways. After incubation
with [3H]ADP to establish an equilibrium the addition of casein to the reaction resulted in
the recovery of lesser amounts of [3H]ADP-Lon complex over time than in the absence of
casein. They also noted that basal ATP hydrolysis was inhibited by a greater percentage
in the absence versus the presence of casein (39). Thomas-Wohlever and Lee confirmed
these observations using steady-state ADP inhibition analyses of ATP-dependent S3
cleavage by Lon protease. They measured Kis and Kii values which reflected affinity of
Lon for ADP in the presence of very low and high levels of S3, respectively. The affinity
for ADP was weakened in the presence of higher levels of S3 (Kii > Kis) presumably by
binding allosterically to promote ADP/ATP exchange (40). Collectively theses results
would suggest that the rate of ADP release is possibly increased in the presence of
peptide or protein substrate. However, the results of the stopped flow MANT-ADP
release experiments summarized in Table 6.6 demonstrate that there is no difference in
182
the off rate in the presence of peptide (Table 6.6). The rate constant for ATP binding and
the conformational change detected upon nucleotide binding were also unaffected by
peptide or protein substrate (Table 6.4). As no pre-steady-state rate constant identified
thus far has been affected by S2 peptide, we concluded that the pre-steady-state rate
constants determined in Chapters 3-6 describe the first round of ATP hydrolysis which is
independent of peptide (Scheme 7.1). If peptide is present, we propose it acts on an
enzyme form following the first round of ATP hydrolysis which is then catalytically
active and described by the peptide stimulated kcat (1 s-1, Table 6.2).
Scheme 7.2 represents the proposed kinetic mechanism for ATPase activity in the
presence of peptide. On the basis that peptide affects only the steady-state ATPase
activity of the low-affinity sites, we propose that Lon adopts a different form after its first
turnover as an ATP-dependent protease, i.e., from IV to VI. Enzyme form VI is
speculative, although the tryptic digest studies described in Chapter 4 tentatively support
its existence because they probed a form of Lon with ATP bound only to the high-affinity
sites which also stabilized the 63 kDa band indicative of the ATP-dependent closed
conformational change (94). Catalytic steady-state turnover can then occur (forms VI –
VIII) as described by the peptide-stimulated kcat = 1 s-1 (Table 6.2). The individual steps
along this pathway will need to be defined in the future, but will depend on the ability to
isolate enzyme form VI to study using pre-steady-state techniques. Peptide could still be
interacting with enzyme form VIII or IV to promote ADP release, which would be
consistent with the observations proposed in the steady-state studies. Additionally as the
conformational change step is relatively slow as well, peptide could be speeding up this
step as well. The details concerning exactly how the peptide is interacting with the
183
Scheme 2
ADP PeptideATP
delivery &
cleavage
koff,1 = 0.5 s-1
ADP
IV
Peptide
ATP
k =?
ADP
k =?
ATP
VI
ATP
ADP
ATP
ATP
kcat = 1 s-1
k =?
VIII
VII
As
As in
in Scheme
Scheme7.1
1, in Form (IV) Lon has hydrolyzed the first round of ATP at the low-affinity
sites
sites.
Following ADP release from the low-affinity ATPase site after the first turnover, If peptide
substrate is present, we propose enzyme forms (VI – VII)
Scheme 7.2 – Revised catalytic mechanism for S2 stimulated ATPase activity.
184
enzyme in this pathway will need to be defined and are shown as “peptide delivery
cleavage” in Scheme 7.2. As described in the results section of this chapter, the
simulation program, FitSim, was used to confirm the plausibility of the revised
mechanism proposed in Scheme 7.2.
In order to test the validity of the newly revised model for Lon catalysis in the
presence of peptide substrate, further experimentation will need to be performed.
Specifically, the individual rate constants associated with the peptide stimulated turnover
of the proposed enzyme form IV will need to be probed including ATP binding,
hydrolysis, and product release (Scheme 7.2). Because the kcat of this process is faster
than the kcat for the intrinsic ATP hydrolysis described by Scheme 7.1, the rate limiting
step or steps must be faster than ADP release from Scheme 7.1. A starting point for
probing the peptide stimulated ATPase mechanism is to measure the steps associated
with the proposed enzyme form IV analogous to the pre-steady-state experiments
described in this thesis which probed free enzyme. As pre-steady-state experiments that
dissect the steps associated with peptide hydrolysis are currently being investigated in
detail by Jessica Patterson-Ward, the timing and coordination of the ATPase and
peptidase activities can be integrated into a comprehensive model for Lon activity. The
ATP-dependent peptidase activity displays a pre-steady-state lag when measuring peptide
hydrolysis (Chapter 3). This indicates that a buildup of a reaction intermediate is
necessary prior to steady-state peptide hydrolysis. The proposed enzyme form IV could
be the intermediate necessary for the initiation of peptidase activity. That is, an enzyme
form capable of ATPase and peptidase turnover is generated subsequent to the first round
of ATP hydrolysis. This hypothesis would predict a decrease or elimination of the lag in
185
peptide hydrolysis if the enzyme form IV is generated prior to monitoring peptidase
activity. Therefore, one could design a double mixing stopped flow experiment to probe
this prediction. Unlabeled peptide, ATP, and Lon could be mixed for a couple of seconds
to generate enzyme form IV prior to the rapid addition of fluorescent peptide. The design
of these types of experiments could lend support to the proposed enzyme form. The
breadth of these experiments may need to span beyond kinetic techniques.
Structure function studies could also provide unique insight into the mechanism.
For example using tryptophan as a probe for intrinsic Lon fluorescence, mutants could be
designed which allow the rates of specific conformational changes such as the proposed
nucleotide dependent conformational change to be measured. In addition, mutations of
the ATP binding domain which prevent ATP binding and hydrolysis could be used to
explore the coupling of the high- and low-affinity ATPase sites. If the seemingly “noncatalytic” ATPase site were eliminated, what affect would that have on Lon activity?
One could design a fused form of Lon which would be expressed as a “dimer” containing
one functional ATPase domain and one non-functional domain to attempt to answer this
question. If this protein were expressed, assembled into the higher order oligomer, and
displayed activity similar to wild type Lon, it could simplify the study of the steps
associated with the peptide-stimulated ATPase reaction.
This chapter describes the preliminary experiments I have done both to isolate
ATP hydrolysis and ADP release from the putative enzyme form IV as well as
preliminary “structure function” studies to measure the rate of the proposed ATP
dependent conformational change. Because the design of Lon mutants used to probe
conformational changes would be greatly aided by the availability of an intact crystal
186
structure of Lon, this chapter also describes the preliminary crystal screening I have
performed. To isolate the first round of ATP hydrolysis by the putative enzyme form IV,
a four syringe rapid quench experiment was designed. It is explained in detail in the
results section. The release of ADP from the putative enzyme form IV was attempted to
be visualized using a double mixing stopped-flow experiment which is also described in
detail in the results section. Furthermore, I designed a tryptophan Lon mutant to measure
the rate of the proposed ATP dependent conformational change. Lastly the initial crystal
screens of E. coli Lon are outlined in the results. The preliminary data described in this
chapter can be used as a starting point for testing the unique mechanism proposed in
Scheme 7.2 as well as expanding the tools used for probing the mechanism beyond
kinetic techniques.
187
Materials and Methods
Materials. Nucleotides were purchased from Sigma or ICN Biomedical. Cloning
reagents were purchased from Promega, New England BioLabs Inc., Invitrogen and USB
Corporations. Oligonucletoides were purchased from Integrated DNA Technologies Inc.
Crystallization Screens I and II and miscellaneous crystal screening tools were purchased
from Hampton Research. [α-32P]ATP was purchased from Perkin Elmer. Buffer
reagents and PEI cellulose TLC plates were purchased from Fisher.
General Methods. Peptide synthesis and protein purification procedures were performed
as described previously (40). Synthesis of MANT-ATP and MANT-ADP was performed
by Xumei Zhang as described previously (108, 110). All enzyme concentrations were
reported as Lon monomer concentrations. All reagents are reported as final
concentrations. Unless otherwise stated all experiments were performed at 37 °C.
Four Syringe Rapid Quench Assays. The acid-quenched time courses for ATP hydrolysis
were measured using a rapid-chemical-quench-flow instrument from KinTek
Corporation. The instrument can be adapted for a four syringe quench experiment by
utilizing the quench syringe for a third reagent, the second quench delay time adjusted for
the individual time points (0 – 4 s), and the constant quench volume of 85.4 µL is used.
The acid quench is then placed in the collection tube to stop the reaction. All solutions
were made in 50 mM HEPES buffer at pH 8.0, 5 mM DTT, 5 mM Mg(OAc)2, and 75
mM KOAc. A 15 µL buffered solution of 30 µM Lon and 2.5 mM S2 peptide was
rapidly mixed with a 15 µL buffered solution of 100 µM ATP at 37 °C for 0.4 s followed
by the addition of a buffered solution of 100 µM [α-32P]ATP for time points varying
between 0 – 4 s. The mixture was then expelled through the exit line and quenched with
188
0.5 N formic acid. A 3 mL aliquot of the aqueous solution was spotted directly onto a
PEI-cellulose TLC plate (10 cm x 20 cm) and the plate developed in 0.75 M KPi buffer
(pH 3.4). The amount of [a-32]ADP produced at each time point was quantified using the
Packard Cyclone storage phosphor screen Phosphor imager purchased from Perkin-Elmer
Life Science. To compensate for slight variations in spotting volume, the concentration
of the ADP product obtained at each time point was corrected for using an internal
reference as shown in eq 1.

ADPdlu
[ ADP] = 
 ATPdlu + ADPdlu

 × [ ATP ]

(1)
All assays were performed at least in triplicate and the average of those traces used for
data analysis. The observed steady-state rate constants (kss,ATP) were determined by
fitting the data with the linear function, Y= mX +C, where X is time, Y is [ADP] / [E], m
is the observed steady-state rate constant in s-1, and C is the y-intercept. Data fitting was
accomplished using the nonlinear regression program KaleidaGraph (Synergy).
Double Mixing Stopped Flow Assays. Pre-steady-state experiments were performed on a
KinTek Stopped Flow controlled by the data collection software Stop Flow version 7.50
β. The sample syringes were maintained at 37 º C by a circulating water bath. All
syringes were buffered in 5 mM Mg(OAc)2, 50 mM HEPES pH 8, 75 mM KOAc, 5 mM
DTT, and 20% glycerol. Syringe A contained 15 µM E. coli Lon monomer pre-incubated
with 15 µM ADP. Syringe B contained 3 mM ADP with and without 1.5 mM S2
peptide. Syringe C contained varying amounts of MANT-ATP (30 - 300 µM). The valve
for syringe C was opened on the KinTek Stopped Flow, and the delay line calibrated at
33 µL resulting in a second push volume of 46 µL. The contents of Syringe A and C
189
were rapidly mixed for times between 2 – 35 s prior to the second push which mixed the
developed reaction with the contents of Syringe B and monitoring the sample cell.
MANT-ADP release was detected by a decrease in fluorescence (excitation 360 nm
emission 400 nm longpass cutoff filter) resulting from rapid mixing of syringe contents in
the sample cell. The resulting exponential data were a result of averaging at least four
traces. All experiments were performed in triplicate and the averaged time courses fit
with equation 3.
(
) (
Y = A1 exp − k1t + C1 + A2 exp − k 2 t + C2
)
(3)
where t is time in seconds, A1 and A2 are amplitudes for the first and second exponential
phases respectively in relative fluorescence units, k1 and k2 are the observed rate
constants for the first and second exponential phases in seconds, and C1, C2 are constants.
Cloning Y461W Mutant. The E. coli Lon gene pJW018 in the pET-24c (+) contained the
mutations W297F, W303F, W603F, and S679W. Using the Stratagene site directed
mutagenesis kit, the forward primer 5’-AAAGATGGTCCGAGTGCCGGTATTGCT-3’,
and the reverse primer 5’-AGCAATACCGGCACTCGGACCATCTTT-3’, the S679W
mutation was restored to the wild type S679. The resulting plasmid named pDV024
contained the mutations W297F, W303F, and S679W. Using the Stratagene site directed
mutagenesis kit, the forward primer 5’CCACTACCTGGAAGTGGATTGGGATCTCAGCGACG-3’, and the reverse primer
CGTCGCTGAGATCCCAATCCACTTCCAGGTAGTGC-3’, the mutation Y461W was
introduced into pDV024. The resulting plasmid was named pDV025. The Y461W
mutation was also engineered into the wild type E. coli Lon gene pHF004 using the
identical primers and site directed mutagenesis kit. The resulting plasmid pDV026
190
contained only the Y461W mutation. pDV026 was overexpressed in BL21(DE3) cells,
selected for with 30 mg/mL kanamycin, and induced at OD600 = 1 with 1mM IPTG for
one hour. The protein was isolated and purified in the same manner as wild type E. coli
Lon.
Stopped Flow Intrinsic Fluorescence Assays. Pre-steady-state experiments were
performed on a KinTek Stopped Flow controlled by the data collection software Stop
Flow version 7.50 β. The sample syringes were maintained at 37 º C by a circulating
water bath. All syringes were buffered in 5 mM Mg(OAc)2, 50 mM HEPES pH 8, 75
mM KOAc, 5 mM DTT, and 20% glycerol. Syringe A contained 5 µM E. coli Lon or
Y461W mutant with and without 500 µM S2. Syringe B contained 1 mM ATP or 1 mM
AMPPNP with and without 500 µM S2 peptide. Changes in intrinsic tryptophan
fluorescence (excitation 290 nm emission 340 nm longpass cutoff filter) were detected as
a decrease in flourescence resulting from rapid mixing of syringe contents in the sample
cell. The resulting exponential data were a result of averaging at least four traces. All
experiments were performed in triplicate and the averaged time courses fit with equation
4.
(
)
Y = A exp − k1t + C
(4)
where t is time in seconds, A is the amplitude for the exponential phase in relative
fluorescence units, k is the observed rate constant for the exponential phases in seconds,
and C is a constant.
Crystal Screening. The ∆-N E. coli Lon mutant was overexpressed in BL21(DE3) cells,
induced, and purified identically to wild type E. coli Lon. The truncated mutant
displayed comparable peptidase and ATPase activity to wild type E. coli Lon. The
191
mutant enzyme was stored in 50 mM HEPES pH 8.0, 50 mM Na2H2PO4, 2 mM DTT,
0.01% tween 20, and 20% glycerol. In Dr. Focco van der Akker’s lab with the assistance
of Pius Padayatti, the grid screens contained 13.5 mg/mL ∆-N Lon (1 µL:1 µL), and were
monitored at 20°C. All other commercially available screens contained 13.5 mg/mL ∆-N
Lon (0.5 µL:0.5 µL), and were monitored at both 20°C and 4°C. In Dr. Irene Lee’s lab
Hampton Research Crystal Screens I and II were monitored at room temperature. ∆-N
Lon was mixed with 250 µM MG262 + 500 µM ATP, 400 µM phenol-indole, or 500 µM
ADP for screening. Any crystals detected in the commercial screens were dyed with 0.5
µL Izit dye to determine if they were salt crystals or protein crystals.
192
Results and Discussion
Kinetic Simulation Matches Proposed Model. Previously, we reported a sequential ATP
hydrolysis reaction model to account for the functional nonequivalency detected in the
two ATPase sites in Lon (Chapter 3) (77). This model assumed that ATP occupancy at
the low-affinity sites promoted the subsequent hydrolysis of ATP at the high-affinity
sites. Further experimentation revealed that the hydrolyses at the two ATPase sites are
independent of one another and that the pre-steady-state burst in ADP production could
be attributed solely to low-affinity ATPase site activity (Chapter 4) (94). In light of the
results obtained in these studies I revised the former kinetic model for Lon ATPase
activity as shown in Scheme 7.3. To check for consistency between the revised model
and the S2-stimulated ATP hydrolysis time courses shown in (Figure 3.6), I collectively
re-fit the data to the revised mechanism by regression analysis using FitSim (114-116).
The results are summarized in Table 7.1 and Figure 7.1. As the burst amplitude but not
the burst rate constants of the time courses varied with [ATP], I input the burst amplitude
values determined previously as the specified enzyme concentration for the
corresponding [ATP] in the fitting process. In Scheme 7.3, the enzyme form F represents
only the low-affinity ATPase sites and corresponds to the graphical enzyme form IV
depicted in Schemes 7.1 and 7.2. It is discernible from Figure 7.1 that the kinetic data
overlay well with the fitted time courses and from Table 7.1 that the theoretical rate
constants agree closely with those obtained experimentally. It should be noted that
although the hydrolytic activities of the high- and low-affinity sites are independent of
one another, it is possible that the binding of ATP to the high-affinity sites may still have
193
E+A
a
k+1
k-1
EA
k+2
k-2
FP
k+3
k-3
F+P
A+F
k+4
k-4
FA
k+5
k+1 = 0.7 µM-1 s-1
k+4 = 10 µM-1 s-1
k-1 = 7 s-1
k-4 = 5 s-1
k+2 = 8 s-1
k+5 = 50 µΜ s-1
k+3 = 0.5 s-1
k+6 = 5 s-1
k-3 = 1 µM-1 s-1
k-6 = 5 µM-1 s-1
k-5
FQ
k+6
k-6
F+Q
E and F are different catalytic forms of Lon along the reaction pathway.
Scheme 7.3 – Mechanism used for the collective fit of the kinetic data using the program
FitSim.a
Table 7.1: Comparison of Experimentally Obtained Rate Constants with those
Determined from the Collective Fit of the Data to the Kinetic Mechanism in Scheme 7.3
K+1
k-1
K+2
K+3
k-3
K+4
k-4
K+5
K+6
k-6
*
#
FitSim
Values
0.7 µM-1 s-1
7 s-1
8 s-1
0.5 s-1
1 µM-1 s-1
10 µM-1 s-1
5 s-1
50 µM s-1
5 s-1
5 µM-1s-1
Experimental
Values
0.7 µM-1 s-1
7 s-1
*
11 s-1
0.5 s-1
0.6 µM-1 s-1
#
ND
#
ND
#
ND
#
ND
#
ND
values obtained from reference (77)
values have yet to be experimentally determined
194
8
7
[ADP], µM
6
5
4
3
2
1
0
0
0.5
1
1.5
2
time (s)
Figure 7.1 - Collective fit of acid-quench ATPase data from reference (77) using FitSim.
Simulation of the ATPase mechanism outlined in Scheme 7.3 was performed using
FitSim. The resulting solid lines yielded the rate constants summarized in Scheme 7.3,
Table 7.1 and were overlayed with the experimental data from reference (77) for ATP
hydrolysis. This demonstrates consistency with the proposed sequential mechanism. The
experimental time courses represent the hydrolysis of α32P[ATP] as determined using an
acid quench experiment at (x) 5 µM ATP, (+) 10 µM ATP, (◊) 25 µM ATP, (V) 50 µM
ATP, (○) 100 µM ATP, and (□) 200 µM ATP (Chapter 3).
195
an effect on ATP binding to the low-affinity sites such that only the burst amplitude but
not the burst rates of ATP hydrolysis at the low-affinity sites are affected. The kinetic
experiments discussed thus far cannot resolve this issue. Methods beyond kinetics are
likely necessary to further address the mechanism.
Probing ATP Hydrolysis by the Putative “F” Form. Schemes 7.1, 7.2, and 7.3 proposed
a revised kinetic mechanism for Lon protease based on the experiments described in the
previous chapters. Integral to the revised mechanism is the proposal of enzyme form IV
(Scheme 7.1, 7.2, called “F” in Scheme 7.3). Enzyme form IV or “F” is a form of Lon
which is proposed to exist following the first round of ATP hydrolysis at the low-affinity
sites and catalyzes steady-state ATPase turnover faster in the presence of peptide or
protein substrate. The next step in experimental design is to isolate the “F” form of Lon
and measure the rate constants associated with its catalysis of ATP hydrolysis in order to
ascertain which step in the reaction has a stimulated rate due to the presence peptide. I
performed some preliminary studies to attempt to isolate the “F” form of Lon. The first
experiment I designed to monitor the first round of hydrolysis by the “F” form
(conversion of VII to VIII, Scheme 7.2). I utilized the simulation program KinSim to
generate an “expected” time course (Figure 7.2) of the first rounds of ATP hydrolysis by
the “F” form using the proposed mechanism shown in Scheme 7.3. The rate constant for
hydrolysis by the “F” form (k+5 = 50 µM s-1) in Scheme 7.3 has not been experimentally
determined. Therefore, I allowed it to vary from 1 – 50 s-1 in the simulation of the
“expected” time courses of the first rounds of ATP hydrolysis by the “F” form (Figure
7.2). The predicted time courses are somewhat linear in nature following 0.4 s. In order
196
70
60
[ADP], µM
50
40
30
20
10
0
0
1
2
3
4
5
time (s)
Figure 7.2 – Simulation of expected time courses of “F” form hydrolysis at 5 µM Lon,
500 µM S2, and 100 µM ATP. The (○) show the expected burst of ATP hydrolysis by
the “E” form. The “F” form hydrolysis is simulated by the other four traces with the rate
constant for hydrolysis varying from 1 s-1 (□), 5 s-1 (◊), 10 s-1 (U), to 50 s-1 (V).
197
to probe this rate constant experimentally, the “F” form would need to first be generated,
and then the hydrolysis was monitored. To accomplish this I designed a four syringe
rapid quench experiment. The theory behind the experiment was to allow the first round
of ATP hydrolysis to occur to generate the “F” form, and then introduce 100 µM [α32
P]ATP to monitor subsequent hydrolysis. The rapid quench experiments performed in
Chapter 3 showed that the first round of ATP hydrolysis by Lon (the “pre-steady-state”)
was essentially complete by 0.4 s. Thus a 0.4 s delay would be required prior to the
addition of [α-32P]ATP. Modifying our three syringe rapid quench for use in a four
syringe quench experiment requires the use of more reagents including [α-32P]ATP, S2
peptide, and Lon. However, if this method proved successful, a four syringe quench box
could be purchased from KinTek to perform experiments with reagent amounts
comparable to the three syringe quench experiments. In order to accomplish this
experiment on the three syringe rapid quench the radiolabeled ATP (100 µM) must be
added to the quench syringe so that the unlabeled ATP (100 µM) and Lon (30 µM) can
first mix for 0.4 s and then be introduced to the radiolabeled substrate (100 µM [α32
P]ATP (Figure 7.3). According to the rapid quench manual, the volume of radiolabeled
ATP coming from the quench line is 85.4 µL, therefore the enzyme reaction is diluted by
approximately six fold resulting in a final Lon concentration of approxiamately 5 µM in
the reaction. However in order to complete a time course the volume of radiolabeled
ATP had to at least be tripled. Therefore, more than 85.4 µL must have been pushed
through the quench syringe for each time point. Figure 7.4 shows the data from the four
syringe quench experiment. The time course was fit with a linear equation to obtain the
steady-state rate constant, 0.13 s-1. This value is lower than the steady-state rate constant
198
100 µM [α32
P] ATP
60 µM Lon +
5 mM S2
0.4 s
100 µM ATP
0-5s
Figure 7.3 – Depiction of the 4-syringe quench reaction to monitor the first round of
turnover by the “F” form.
3.5
2.5
2
32
[α- P]ADP (µM)
3
1.5
1
0.5
0
0
1
2
3
4
5
time (s)
Figure 7.4 – Four syringe rapid quench time course of ATP (100 µM) hydrolysis by 5
µM Lon in the presence of 500 µM S2. Lon was rapidly mixed with 100 µM ATP for 0.4
s in an attempt to generate the “F” enzyme form prior to the addition of 100 µM [α32
P]ATP. The data was fit with a linear equation to obtain the steady-state rate constant,
0.13 s-1.
199
for S2-stimulated hydrolysis of 100 µM ATP (0.6 s-1). One possible explanation for the
discrepancy is that the final concentration of enzyme is not precisely known due to the
dilution from the [α-32P]ATP from the quench line. Furthermore, because the initial
concentration of protein was very high, a precipitate was visually obvious in the reactions
with casein. If the peptide, casein, or some of Lon were precipitating the lower kcat value
would be expected. The same experiment attempted using the four-syringe box, where
the volumes of the reactants can be calibrated, would reconcile this question. Another
option would be to calibrate the quench volume using scintillation counting similar to
what was described in Chapter 3.
The four syringe quench experiment needs to be optimized in general including
the concentrations of Lon, S2, and ATP to improve the quality of the data to ensure that
the time course is indeed linear. Increasing the concentration of cold ATP would ensure
that all Lon is bound to ATP during the generation of the “F” form (0.4 s-1) so that no free
enzyme exists when the radiolabeled ATP is introduced. The initial region of the time
course in Figure 7.4 (0-0.6 s) appears to possibly have a small burst in activity. If free
Lon were in the solution because 100 µM ATP was not enough to drive 30 µM Lon to
bind, then the small burst could be a result of the minute population of free enzyme
hydrolyzing radiolabeled ATP. Increasing the concentration of Lon in the reaction could
improve the quality of the data by ensuring that more product signal is generated.
Furthermore, more time points could be added to define the initial portion of the time
course.
If the first round of ATP hydrolysis does indeed generate the “F” form, it is
possible that no typical pre-steady-state burst in ATP hydrolysis would ever be detected
200
from the “F” form because it may never exist unbound to nucleotide. The binding and
hydrolysis of ATP by the “F” form would thus be limited by the release of ADP and Pi
generated from the first turnover of ATP hydrolysis by the “E” form. So, monitoring the
hydrolysis of the “F” form of Lon may always mirror a linear steady-state turnover even
on the millisecond time scale. If this is the case, reaction steps in the “F” form S2
stimulated ATP hydrolysis pathway other than hydrolysis could be monitored. The linear
time course from the four-syringe quench experiment shown in Figure 7.4 is supportive
of the “F” form being generated prior to ADP release from the first round of turnover as
proposed in Scheme 7.3 (EA→FP). However the experiment does not conclusively
identify a rate constant with the first round of “F” form ATP hydrolysis.
Monitoring MANT-ADP Release from the Putative “F”. If ADP release from the lowaffinity site is the rate limiting step in the “F” form pathway, and if MANT-ADP release
could be detected from the “F” form, it would be expected to be approximately 1 s-1
which is the kcat describing S2 stimulated ATP hydrolysis. Therefore preliminary double
mixing experiments were employed on the stopped flow instrument to isolate ADP
release from the “F” form. If our hypothesis is correct, the presence of S2 peptide should
increase the rate constant associated with MANT-ADP release from the “F” form. A
double mixing experiment described in detail in Materials and Methods was performed.
The concentration of the reagents is diluted by three fold throughout a double mixing
experiment so the initial concentrations in the syringes must account for the dilutions. 15
µM Lon was rapidly mixed with 300 µM MANT-ATP for times varying from 2 – 35 s,
and then the fluorescent nucleotide was competed off of the enzyme by the addition of 3
mM ADP in the presence and absence of 1.5 mM S2 peptide. The fluorescent signal
201
generated from this experiment (excitation 360 nm, 400 nm longpass cut off filter) is
ambiguous because a decrease in fluorescence must be assumed to occur from both
MANT-ATP and MANT-ADP release from both the high- and low-affinity ATPase sites,
as well as both the “E” and “F” enzyme forms because the trigger for their conversion is
not fully understood. In order to simplify the fluorescent signal, 15 µM ADP was preincubated with 15 µM Lon in order to “block” the high-affinity ATPase sites similar to
the technique used in Chapter 4. Figure 7.5 shows time courses of MANT-Nu release
from both free Lon and Lon with its high-affinity sites “blocked” with ADP. However,
the possibility of ADP binding to the low-affinity sites under these conditions cannot be
excluded. All time courses were fit using a double exponential equation and the kinetic
constants are summarized in Table 7.2. The approximate 6 fold increase in amplitude
rather than the expected ~2 fold increase in amplitude from the free enzyme traces shown
in Figure 7.5 (“both sites”) compared to the traces where the high-affinity sites are
blocked with ADP (“low-affinity sites”) confirms that some of the low-affinity ATPase
sites must also be blocked under those conditions. The time delay allowing for the
hydrolysis of MANT-ATP to MANT-ADP was varied from (2 – 35 s) in an attempt to
find an optimal time which all allowed for the generation of the “F” form while balancing
other factors such as the amount of MANT-ATP hydrolysis that occurs and reducing time
for exchange of MANT-ATP and MANT-ADP. Thus varying amounts of free MANTATP, free MANT-ADP, Lon:MANT-ADP, or Lon:MANT-ADP/MANT-ATP must be
present at the start of the experiment due to the varying delay times. These amounts
would presumably vary depending of the initial concentration of MANT-ATP as well.
202
0
"low-affinity sites"
relative fluorescence
-0.5
-1
-1.5
-2
-2.5
"both sites"
-3
-3.5
0
5
10
15
20
25
30
time (s)
Figure 7.5 – Representative time courses of MANT-ADP release in double mixing
experiments in the presence (□, x) and absence (○,◊) of 500 µM S2 peptide. The “lowaffinity” time courses utilized the ADP “blocking” technique at the high-affinity sites,
whereas the “both sites” time courses utilized free Lon. As expected the amplitudes are
greater when MANT-Nu was being released from both sites. The time courses were fit
using equation 3. Rate constants from the double mixing experiments are summarized in
Table 7.2.
Table 7.2: Summary of MANT-Nu Off Rates from “Blocked” or Free Lon in Double
Mixing Experiments
Enzyme form
Lon:ADP
Lon:ADP + S2
Lon:ADP
Lon:ADP + S2
Lon:ADP
Lon:ADP + S2
Lon
Lon + S2
delay time
(s)
35
35
10
10
2
2
10
10
[MANT-ATP]
(µM)
100
100
100
100
10
10
10
10
203
koff,1
(s-1)
1.0 + 0.1
0.8 + 0.1
0.7 + 0.1
0.8 + 0.1
1.4 + 0.2
1.0 + 0.1
0.9 + 0.1
0.9 + 0.1
koff,2
(s-1)
0.3 + 0.1
0.2 + 0.1
0.2 + 0.1
0.2 + 0.1
0.3 + 0.1
0.2 + 0.1
0.2 + 0.1
0.2 + 0.1
A1
(cps)
0.13 + 0.01
0.17 + 0.01
0.11 + 0.01
0.11 + 0.01
0.32 + 0.01
0.52 + 0.01
1.8 + 0.01
1.8 + 0.01
A2
(cps)
0.11 + 0.01
0.07 + 0.01
0.03 + 0.01
0.03 + 0.01
0.35 + 0.01
0.16 + 0.01
1.0 + 0.01
1.3 + 0.01
As we were attempting to detect an effect on the MANT-ADP release rate
constant from peptide, the experiments were performed in the presence and absence of
peptide so that the results could be compared. When peptide was added to the reaction,
the same two rate constants were always detected (Table 7.2) regardless of the “blocking”
technique, varying delay times, or initial MANT-ATP concentration. It is possible that
the two numbers describe MANT-ADP and MANT-ATP release from the low-affinity
site, but the possibility that the two rate constants are from the release of MANT-ADP
from the low-affinity site of the “E” and “F” form cannot be excluded. The numbers do
correlate with the two kcat values for ATP hydrolysis (0.5 s-1 and 1 s-1) however unlike the
kcat values these observed rate constants (Table 7.2) are independent of the presence of
peptide. Furthermore, it is not clear what the kobs = 1 s-1 (Table 7.2) would be describing
in the intrinsic pathway. The amplitude of the release of MANT-Nu from “both sites” in
the presence of peptide is greater than in the absence of peptide (Figure 7.5). This
suggests that S2 peptide is possibly promoting the release of ADP.
The fluorescence signal from this type of experiment will need to be dissected
further in order to conclusively assign the observed off rates to specific events. This will
likely only be accomplished by identifying new ways to eliminate fluorescent signal from
the possible places (MANT-ATP and MANT-ADP release from both the high- and lowaffinity sites). One could attempt to block the high-affinity sites by covalently modifying
their active sites and then perform the experiment similar to that described above. This
would eliminate fluorescent signal from the high-affinity sites, but not from the two
nucleotides. Adjusting the delay time and the concentration of MANT-ATP to ensure
that all was hydrolyzed to MANT-ADP would eliminate the fluorescent signal from the
204
two nucleotides. Although this experiment did not result in conclusive data for release of
ADP from the “F” form, these double mixing stopped flow experiments could be further
explored to isolate an increase in the rate of MANT-ADP release from the “F” form in
the presence of S2 peptide using the suggestions outlined above. It is also possible that
peptide does not increase the rate of ADP release. As discussed in the introduction of
this chapter the hypothesis was based on the observations such as the increase in Ki for
ADP in the presence of peptide. As Ki is a function of both the rate of ADP binding and
release it is possible that the on rate of ADP to the “F” form is affected rather than the off
rate. It is also feasible that a conformational change step in “F” turnover which results in
the increase in steady-state rate. We could also attempt a similar double mixing
experiment to probe whether the rate constant for the putative conformational change
associated with nucleotide binding is increased. In addition a structure/function approach
to investigating the mechanism more steps in the pathway could be identified and probed.
Design of Tryptophan Mutants of Lon to Probe Conformational Changes. One approach
to probing the catalytic mechanism of Lon in more detail as described above is to design
mutants that will enable the detection of various steps along the pathway including
conformational changes. Because no structure of intact Lon protease is available, this
task involves many assumptions. As discussed in Chapter 2 the structure of HslUV, the
bacterial homolog of the proteasome, is used as a model for Lon because they are both
members of the AAA+ family of enzymes. One notable difference between the two
enzymes is that HslU, the ATPase domain, is a separate subunit from HslV, the protease
domain. The greatest homology between Lon and HslUV lies between HslU and the
ATPase domain in Lon (25, 26). The tryptic digest studies discussed in Chapter 2 as well
205
as the stopped flow binding studies of MANT-ATP discussed in Chapter 6 suggested a
nucleotide dependent conformational change in Lon. This conformational change
protected Lon from tryptic digestion which indicated that perhaps the enzyme was
adopting a closed conformation rendering cleavage sites inaccessible to trypsin (55). The
MANT-ATP stopped flow binding studies revealed a rate constant, kon,2 ~ 5 s-1, which
was independent of nucleotide concentration. This was also indicative of a
conformational change however both of these methods were indirect. Therefore in order
to directly monitor the rate of the conformational change, I wanted to engineer a
trytophan residue in Lon whose fluorescence would be directly affected by the proposed
nucleotide dependent conformational change. As shown in Figure 7.6, HslU adopts a
more open conformation when bound to ATP in order to accommodate the γ-phosphate
group and any metal bound to the ATP (61). Using the program SIM (expasy.org), I
performed a sequence alignment of HslU (117) with the ATPase domain of E. coli Lon
protease to determine if similar helical regions existed in Lon. There was a region
containing 56% sequence identity which corresponded to the α-helical portion of HslU
that rotates in the different nucleotide induced conformations (Figure 7.6). In this region
in Lon there is a conserved tyrosine residue (Y461). In order to attempt to detect a
nucleotide dependent conformational change by fluorescence, I utilized site directed
mutagenesis to engineer Y461W. Figure 7.7a shows the SDS-PAGE of the tryptic digest
comparison of wild type Lon and Y461W visualized by Coomassie Brilliant Blue stain.
The wild type and mutant enzyme display similar digestion patterns in the presence and
absence of S2 peptide. Figure 7.7b reveals that Y461W Lon retains comparable
206
Figure 7.6 – There are four distinct HslU conformational states. They are the
ADP/dADP (silver), ATP (cyan), SO4 (magenta), and empty (orange) states. The αhelical domain rotates as a rigid body along a single axis near Box VII’ marked by the X.
The rotation resulting from the various states are listed with respect to the ADP/dADP
conformational state. Adapted from Wang, et al. (61).
207
a Time (min):
0 20 40 0 20 40 0 20 40 0 20 40
Y461W
Y461W
+ S2
WT
WT + S2
b
5
1.25 10
5
relative fluorescence
1.5 10
1 10
5
7.5 10
4
5 10
4
2.5 10
4
0
0
50
100
150
200
250
300
time (s)
Figure 7.7 – Characterization of Y461W E. coli Lon mutant. (a) SDS-PAGE visualized
by Coomassie brilliant blue shows 5 µM Y461W and WT Lon in the presence and
absence of 500 µM S2 peptide digested with a limiting amount of trypsin and quenched
with SBTI at the indicated times. Lane 1 shows the molecular markers in kilodaltons
(from top to bottom): 183,114, 81, 64, 50, 37, 26, 20. (b) Time course of degradation of
S3 peptide by WT (□) and Y461W (○) E. coli Lon (excitation 320 nm, emission 420 nm).
208
peptidase activity to wild type Lon. As Y461W Lon seemed to be a reasonable analog of
wild type Lon, stopped flow experiments were employed to monitor any changes in
intrinsic tryptophan fluorescence (excitation 290 nm, emission 340 nm cutoff filter) in
Y461W Lon. Figure 7.8 illustrates that an intrinsic change in fluorescence occurs only in
wild type Lon when mixed with ATP and S2 peptide. It is interesting to note that the
substitution of a single tyrosine residue altered the intrinsic fluorescence changes
associated with wild type Lon. One possibility for this is that Y461 was involved with
resonance energy transfer (RET) with one of the Trp residues in Lon, and when it was
mutated the RET was disturbed (54). There are three native tryptophan residues in wild
type Lon (W297, W303, and W603) which are responsible for the fluorescent signal in
wild type Lon. It is likely that the fluorescence from the native tryptophan residues could
interfere with the signal from Y461W. Therefore, an E. coli Lon mutant lacking the three
native tryptophan residues by conservatively mutating them to phenylalanine (pDV024,
W297F, W303F, W603F) could be used as a starting point for the site directed
mutagenesis of Y461W instead of wild type Lon. A Lon mutant similar to this
containing no native tryptophan residues and an additional S679W mutation was made by
Jessica Patterson-Ward. The stability of the nucleotide dependent closed conformation in
this Lon mutant detected by tryptic digest studies was greatly decreased. However, this
Lon mutant was proteolytically inactive so it may be beneficial to express and purify the
“trp-less” Lon mutant (pDV024) which retains the active site serine. It may be display
behavior more comparable to wild type Lon. Then the intrinsic fluorescent changes of
the Y461W mutant could be explored without the interference of the native tryptophan
residues (pDV025).
209
0.1
relative fluorescence
0
-0.1
-0.2
-0.3
-0.4
-0.5
0
0.2
0.4
0.6
0.8
1
time (s)
Figure 7.8 – Stopped flow experiments monitor changes in tryptophan fluorescence
(excitation 290 nm, emission 340 nm longpass cutoff filter) when Y461W is mixed with
ATP and S2 peptide (○) or ATP alone (□). These traces were compared to wild type Lon
mixed with ATP and S2 peptide (◊) or ATP alone (U).
210
Crytal Screens for Lon Protease. Because a crystal structure of intact Lon would
increase the success rate of structure function studies such as the one described above,
crystallization conditions were screened. The N-terminal region of Lon is thought to be
the most fluid part of the enzyme, and is believed to be responsible for the inability to
crystallize wild type Lon. Therefore, a ∆-N mutant of E. coli Lon was constructed for
use in crystallization screens. The ∆-N mutant was overexpressed in BL21 (DE3) and
purified to near homogeneity identically to wild type Lon (Figure 7.9). This mutant (60
kDa) is identical in sequence to the 67 kDa band shown in the tryptic digest studies
whose molecular weight was assigned visually by reference to the molecular weight
marker on the gel. ∆-N Lon is also stabilized from tryptic digestion in the presence of
nucleotide. As shown in Figure 7.10 the ∆-N mutant of E. coli Lon displays peptidase
activity comparable to wild type E. coli Lon. Thus the deletion of the N-terminal region
in ∆-N Lon (- 240 a.a.) did not affect the kinetic constants associated with ATPase or
peptidase activity of the enzyme. This contradicts published data which indicates that the
protease activity of another ∆-N Lon (- 107 a.a.) mutant was drastically reduced (118).
We chose to pursue crystallization of ∆-N Lon in identical buffer conditions to the kinetic
assays performed in our lab because the enzyme is known to be stable and active under
these conditions. Therefore, ∆-N Lon was stored in 50 mM HEPES at pH 8, 50 mM
NaH2PO4, 2 mM DTT, 20% glycerol and 0.01% Tween 20. These conditions are not
ideal for crystallization for a number of reasons. Because the enzyme is buffered so well,
many of the pH adjustments screened for will not be attained, and the high level of salt
will result in many false positives because the salt can also form crystals. For these
reasons a buffer condition containing less salt and buffer was screened. Unfortunately,
211
∆-N E. coli Lon mutant
Wild type E. coli Lon
Molecular weight marker
relative fluorescence
Figure 7.9 - SDS-PAGE of ~540 ng of purified recombinant wild type E. coli Lon (87
kDa) and ~365 ng of ∆-N E. coli Lon mutant (60 kDa) visualized by Coomassie staining.
1 10
5
8 10
4
6 10
4
4 10
4
2 10
4
0
0
50
100
150
200
250
300
time (s)
Figure 7.10 – Degradation of S3 peptide by Lon monitored on Fluoromax 3
spectrofluorimeter (excitation 320 nm, emission 420 nm). ∆-N E. coli Lon displays no
peptidase activity in the absence of ATP (◊), and degrades S3 peptide in the presence of
ATP (□) comparably to wild type E. coli Lon (○).
212
dialysis into 10 mM HEPES at pH 8, 2 mM DTT, 5% glycerol, and 0.01% Tween 20
resulted in complete loss of peptidase activity. The high buffer and salt were necessary
for Lon stability so crystal screens were pursued in spite of the non-ideal buffer
conditions.
I established a collaboration with Dr. Focco van den Akker’s lab in the
biochemistry department for learning basic crystal screening techniques. Both
commercial and self-made crystal screens were utilized as well as temperature conditions
including 20 ° C and 4 ° C. The screens were set up and the wells monitored for crystal
formation for weeks following. Table 7.3 outlines the various crystal screens that were
tested using ∆-N E. coli Lon stored in 50 mM HEPES at pH 8, 50 mM NaH2PO4, 2 mM
DTT, 20% glycerol, and 0.01% Tween 20. All screens were performed in sitting drop
plates, where the enzyme was first mixed with equal amounts of the mother liquor, and
equilibrium was established through vapor diffusion. The commercial screens each
scanned 50-100 crystallization conditions, and the grid screens scanned approximately
24. Any initial hits were double checked for reproducibility by setting up more drops
under the same conditions as well as setting up drops using only the enzyme storage
buffer to screen for salt crystals. If the same type of crystals formed in the buffer only
drops, then the initial hit was most likely salt. Izit crystal dye which is a blue dye that
diffuses into protein channels was also used to determine if initial hits were protein or salt
crystals. However none of the controls can definitively determine whether a crystal is
salt or protein. The presence of protein sometimes aids in the crystallization of salt, and
if the Izit dye does not always penetrate protein crystals. Salt crystals diffract quite
differently than protein, so mounting the crystal and putting it in the beam will
213
Table 7.3: Summary of Crystal Screens Set up in van den Akker Lab
N/A
Temp
(°C)
20
[∆-N Lon]
(mg/mL)
13.5
Drop Size
(µL)
1:1
Initial Hits
(condition #)
None
N/A
20
13.5
1:1
None
Hampton
Research
Hampton
Research
Hampton
Research
Hampton
Research
Hampton
Research
Hampton
Research
Hampton
Research
Emerald
BioSystems
Emerald
BioSystems
Emerald
BioSystems
Emerald
BioSystems
20
13.5
0.5:0.5
7, 16, 17, 18, 22, 24
4
13.5
0.5:0.5
21, 24, 46
20
13.5
0.5:0.5
None
4
13.5
0.5:0.5
21, 37
20
13.5
0.5:0.5
None
4
13.5
0.5:0.5
None
20
13.5
0.5:0.5
48, 81
20
13.5
0.5:0.5
24, 30
4
13.5
0.5:0.5
32, 40, 44
20
13.5
0.5:0.5
11, 29
4
13.5
0.5:0.5
30
Company
PEG 8000 Grid
Screen
Ammonium
Sulfate Grid
Screen
Crystal Screen I
Crystal Screen I
Crystal Screen II
Crystal Screen II
PEG/ION Screen
PEG/ION Screen
Index Screen
Wizard I
Wizard I
Wizard II
Wizard II
214
definitively determine whether the crystal is salt or protein. Because controls could not
distinguish protein crystals from all initial hits, crystals from Crystal Screen I conditions
18 and 22 were sent to the synchrotron. Unfortunately, no initial hits from all of the
screens turned out to be protein. Expansion screens of condition 18 from Crystal Screen I
were also set up because the initial hits seemed promising. No protein crystals were
found in the expansion screen either. Most likely ∆-N Lon enzyme storage buffer
conditions will need to be adjusted or additives such as inhibitors will need to be added to
the screening process.
In order to simplify the initial crystal screening process, we purchased basic
necessities for crystal screening to use in our lab including Crystal Screens I and II from
Hampton Research. I then screened ∆-N Lon in the presence of the known inhibitors
shown in Figure 7.11: MG262 + ATP (21), ADP (1, 40), and 5-phenol-indole at room
temperature. Table 7.4 outlines the initial hits from these crystal screens. All initial hits
were dyed with Izit crystal dye, buffer only drops were set up, and multiple drops were
set up to determine reproducibility. No crystals turned out to be protein. Figure 7.12
shows an example of the types of salt crystals that were found in the crystal screens. This
type of crystal screening will need to be pursued using different types of Lon including
wild type E. coli, Salmonella Typhimurium, human Lon, and other mutants in order to
exhaust all possibilities. Enzyme storage buffer conditions that contain less salt and
buffer will also need to be screened. Obtaining a crystal structure of intact Lon protease
will enhance future structure-function studies of the enzyme which will provide more
insight into the mechanism of this unique enzyme.
215
O
O
H
N
N
H
N
H
B
OH
MG262
OH
NH2
N
O
-
O
P
N
O
-O
O-
P
O
-
O
P
O-
O
O
P
O-
H
H
OH
H
P
H
N
O
O
O
ADP
N
O
OH
O
N
5-Ph-Indolea
O
OH
H
H
OH
H
H
Figure 7.11 – Structures of the Lon inhibitors used in the crystal screening. a Lon
ATPase inhibition data by 5-Ph-Indole was obtained through a personal communication
with the Berdis Lab, Pharmacology Department, Case Western Reserve University.
Table 7.4: Summary of Crystal Screens Performed with Inhibitors
Crystal Screen I
Crystal Screen I
Crystal Screen I
Crystal Screen II
Crystal Screen II
Crystal Screen II
Drop Size
(µL)
Inhibitor
Initial Hits
(condition #)
25
[∆-N
Lon]
(mg/mL)
12.5
0.5:0.5
16, 37, 45
25
11.3
0.5:0.5
MG262 +
ATP
Ph-Indole
25
13
0.5:0.5
ADP
25
12.5
0.5:0.5
25
11.3
0.5:0.5
MG262 +
ATP
Ph-Indole
3, 15, 23,
24, 36, 49
19, 41, 43,
45, 46
2, 5
25
13
0.5:0.5
ADP
29
Company
Temp
(°C)
Hampton
Research
Hampton
Research
Hampton
Research
Hampton
Research
Hampton
Research
Hampton
Research
216
5, 32
Figure 7.12 – Picture of the salt crystals from Hampton Research Crystal Screen I
condition 18.
217
In summary preliminary experiments were performed to explore the steps
associated with ATP hydrolysis by the “F” form of Lon. Although the rate constant
associated with the ATP hydrolysis and ADP release by the “F” form were not
conclusively determined, they do provide a starting point for the design of future studies.
In addition to completing the characterization of the ATPase activity, the details
surrounding the peptidase activity of Lon including substrate specificity and the delivery
of peptide to the active site are being explored by Jessica Patterson-Ward. Once the
mechanism of E. coli Lon is defined, it can be used as a model for the other Lon
homologes such human and Salmonella. In the future differences in substrate specificity
and mechanism between the various homologues can be exploited in inhibitor design.
Other than optimizing specific inhibitors for drug design, they could be used to further
study the function of Lon in vivo.
218
APPENDIX A
LIST OF EQUATIONS
Standardization of Radioactive Data - To compensate for slight variations in spotting
volume on the PEI-cellulose TLC plates in the radioactive assays, the concentration of
NDP product obtained at each time point was corrected for by using an internal reference
as shown below.


NDPdlu
 × [ NTP ]
[ NDP ] = 
+
NTP
NDP
dlu
dlu 

where [NDP] is the concentration of the nucleotide diphosphate, [NTP], is the
concentration of the nucleotide triphosphate, NDPdlu are the density light units
corresponding to the nucleotide diphosphate, and NTPdlu are the density light units
corresponding to the nucleotide triphosphate.
Michaelis Menten Equation – used to extract kcat and Michaelis constant Km from
hyperbolic plot of kobs vs. [substrate] (typically used for Lon NTPase activity)
k obs =
k obs , max [ S ]
K m + [S ]
where kobs is the observed rate constant, kobs,max is the maximal rate constant (equivalent
to kcat), [S] is the concentration of substrate, and Km is the Michaelis-Menten constant
equal to the concentration of S required to reach one half the maximal rate constant.
Hill Equation – used to extract kcat, Michaelis constant Km, and the Hill coefficient n
from the sigmoidal plot of kobs vs. [substrate] (typically used to describe Lon peptidase
activity)
k max [S ]
n
K ' + [S ]
n
k obs =
where kobs is the observed rate constant being measured in units of s-1, kmax is the
maximum rate constant referred to as kss,S3 in units of s-1 (equivalent to kcat), [S] is the
variable peptide substrate in units of µM, K' is the Michaelis constant for S, and n is the
Hill coefficient describing the degree of cooperativity. The Ks (µM) is calculated from
the relationship log K' = n log Ks, where Ks is the [S] require to obtain 50% of the
maximal rate constant of the reaction referred to as Km,ATP or Km,MANT-ATP.
219
Burst Equation – used to extract the burst rate constant kburst, the burst amplitude A, and
the steady state rate constant kss,ATP from the pre-steady-state ATPase time courses.
[ ADP ] /[ E0 ] = A * (1 − e − k burst *t ) + k ss , ATP * t
where [E0] is the concentration of Lon monomer, A is the burst amplitude, kburst is the
burst rate constant, t is time, and kss,ATP is the steady-state rate constant for ATP
hydrolysis.
This equation relies on the rapid accumulation of the Lon:ATP complex meaning
that substrate binding should not limit the reaction under the experimental conditions
used. The burst amplitude for the Lon pre-steady-state ATPase reaction is unusual
because it only approaches half of the enzyme concentration. In addition there is a
transition period between 0.5 – 1 s before the steady-state activity is reached. For
simplicity Figure 3.4 shows only the formic acid quenched S2 stimulated ATPase activity
and outlines the unusual regions in the time course (see below). The black line shows
the fit of data with the standard burst equation. This equation obviously does not
converge well most likely due to the transition phase between the burst and steady-state
activities. Because the pre-steady-state burst activity of Lon is not standard and thus not
described by the burst equation (3), the data was instead split into a single exponential
pre-steady-state phase up to 0.6 s, and a linear steady-state phase for fitting purposes.
4
1
3.5
[ADP] / active site
( µM)
0.8
0.6
[ADP] / active site
( µM)
3
0.4
0.2
2.5
0
2
0
0.2
0.4
0.6
time (s)
0.8
1
Steady-state
1.5
1 burst
0.5
transition
0
0
0.5
1
1.5
2
2.5
3
3.5
time (s)
Figure 3.4 – Pre-steady-state S2 stimulated ATPase activity of E. coli Lon Protease. The
black line shows the data fitting with the burst equation (3). The burst (0 – 0.5 s),
transition (0.5 – 1 s), and steady state phases (1 – 3 s) of the time course are indicated.
The inset zooms in to show the burst and transition phases more clearly.
220
Single Exponential Equation - The burst amplitudes and burst rates were determined by
fitting the kobs data from 0 to 400 ms with the following single exponential equation.
Y = A * exp − k burst t + C
where t is time in seconds, Y is [ADP] in µM, A is the burst amplitude in µM, kburst is the
burst rate constant in s-1, and C is the end point. The observe steady-state rate constants
(kss,ATP) were determined by fitting the data from 600 ms to 1.8 s with the linear function,
Y= mX +C, where X is time, Y is [ADP] / [E], m is the observed steady-state rate
constant in s-1, and C is the y-intercept.
The kinetic parameters from the single turnover experiments described in Chapter 4 were
determined by fitting the time course data with a single exponential equation below.
Y = A * exp − k obs t + C
where t is time in seconds, Y is [ADP] in µM, A is the amplitude in µM, kobs is the
observed rate constant in s-1, and C is the end point.
The kinetic parameters for the double mixing MANT-ADP release experiments which
isolated the high-affinity site were determined by fitting the time course data with a
single exponential equation.
Y = A1 exp − k1t + C1
where t is time in seconds, A1 is the amplitude in relative fluorescence units, k1 is the
observed rate constant for the exponential phase in s-1, and C1 is a constant.
Double Exponential Equation – A double exponential equation (shown below),
preferentially converged over a single exponential equation on the MANT-Nu binding
and release time courses.
(
) (
Y = A1 exp − k1t + C1 + A2 exp − k 2 t + C2
)
where t is time in seconds, A1 and A2 are amplitudes for the first and second exponential
phases respectively in relative fluorescence units, k1 and k2 are the observed rate
constants for the first and second exponential phases in seconds, and C1, C2 are constants.
The meaning of the rate constants describing the two phases had to be determined from
further experimentation (Chapter 6).
Kd Equations from Double Filter Binding Assay – The Kd values for the high- and lowaffinity ATPase sites were probed with a double filter binding assay (Chapter 4). The
radioactive counts at each spot on the membranes were quantified by PhosphorImaging
221
using the Packard Cyclone storage phosphor system. The concentration of bound was
determined according to the equation below,


NCdlu
 * [α 32 P − ATP ]
[bound ] = 
+
 NCdlu + NY dlu 
where NCdlu are the radioactive counts on the nitrocellulose membrane, NY+dlu are the
radioactive counts on the Immobilon Ny+ membrane, and [bound] corresponds to [RL] in
the following equations. The ratio was used in order to eliminate discrepancies in the
numbers due to small errors in spotting volume.
The binding parameters were then determined by fitting the data with the equation below.
[ RL] =
([ R] + [ L] + K d ) − ([ R] + [ L] + K d )2 − 4[ R][ L]
2[ L]
where [L] is the concentration of α32P-ATP, [R] is the concentration of Lon, [RL] is the
concentration of α32P-ATP bound to Lon, and Kd is the equilibrium dissociation constant
for ATP bound at the high-affinity site.
When both the high- and low-affinity sites were probed, the data was fit with the equation
below, which accounted for a tighter binding as well as a weaker binding interaction.
[ RL] =
([ R] + [ L] + K d ) − ([ R] + [ L] + K d ,1 )2 − 4[ R][ L]
2[ R]
+
Bmax, 2 [ R]
K d , 2 + [ R]
where [L] is the concentration of α32P[ATP], [R] is the concentration of Lon, [RL] is the
concentration of α32P-ATP bound to Lon, Kd,1 is the equilibrium dissociation constant for
ATP bound at the high-affinity site, Bmax is the maximal bound complex detected, and
Kd,2 is the equilibrium dissociation constant for ATP bound at the low-affinity site.
222
APPENDIX B
FIRST AUTHOR PUBLICATIONS
223
11432
Biochemistry 2006, 45, 11432-11443
Transient Kinetic Experiments Demonstrate the Existence of a Unique Catalytic
Enzyme Form in the Peptide-Stimulated ATPase Mechanism of Escherichia coli
Lon Protease†
Diana Vineyard, Xuemei Zhang, and Irene Lee*
Department of Chemistry, Case Western ReserVe UniVersity, CleVeland, Ohio 44106
ReceiVed April 25, 2006; ReVised Manuscript ReceiVed July 12, 2006
ABSTRACT: Lon is an oligomeric serine protease whose proteolytic activity is mediated by ATP hydrolysis.
Although each monomeric subunit has an identical sequence, Lon contains two types of ATPase sites
that hydrolyze ATP at drastically different rates. The catalytic low-affinity sites display pre-steady-state
burst kinetics and hydrolyze ATP prior to peptide cleavage. The high-affinity sites are able to hydrolyze
ATP at a very slow rate. By utilizing the differing Kd’s, the high-affinity site can be blocked with unlabeled
nucleotide while the activity at the low-affinity site is monitored. Little kinetic data are available that
describe microscopic events along the reaction pathway of Lon. In this study we utilize MANT-ATP, a
fluorescent analogue of ATP, to monitor the rate constants for binding of ATP as well as the release of
ADP from Escherichia coli Lon protease. All of the adenine nucleotides tested bound to Lon on the order
of 105 M-1 s-1, and the previously proposed conformational change associated with nucleotide binding
was also detected. On the basis of the data obtained in this study we propose that the rate of ADP release
is slightly different for the two ATPase sites. As the model peptide substrate [S2; YRGITCSGRQK(Bz)]
[Thomas-Wohlever, J., and Lee, I. (2002) Biochemistry 41, 9418-9425] or the protein substrate casein
affects only the steady-state ATPase activity of the low-affinity sites, we propose that Lon adopts a different
form after its first turnover as an ATP-dependent protease. Based on the obtained rate constants, a revised
kinetic model is presented for ATPase activity in Lon protease in both the absence and presence of the
model peptide substrate (S2).
Lon protease belongs to the AAA+ superfamily of
ATPases because, like the other members (ClpXP, ClpAP,
ClpCP, HslUV), its proteolytic activity is mediated by ATP
hydrolysis (1-10). This family is based on multiple sequence
alignments which define a common ATPase module and
encompasses a broader range of proteins than the traditional
AAA proteins (ATPases associated with diverse cellular
activities) which are a subfamily of the Walker-type NTPases
(11, 12). Lon protease is distributed throughout the cytosol
in prokaryotic cells while in eukaryotic cells it is localized
to the mitochondria (5, 8, 13). Unlike the other members of
the superfamily which consist of separate regulatory and
proteolytic subunits, the lon gene encodes a single polypeptide subunit containing both the protease and ATPase
domains (12). The subunits in Lon are organized into an
oligomer shown in recently published partial crystal structures as a hexamer (14, 15). The oligomerization of Lon
results in a ring formation with a central cavity and is not
dependent on ATP binding or hydrolysis (16, 17).
Aside from the general interest surrounding Lon due to
its unique coordination of ATPase activity with proteolytic
function, Lon has been shown to be important in maintaining
the virulence of various strains of pathogenic bacteria
including Salmonella enterica serovar Typhimurium (18†
This work was supported by NIH Grant GM067172.
* Corresponding author. Phone: 216-368-6001. E-mail: Irene.lee@
case.edu. Fax: 216-368-3006.
20), Brucella abortus (21), and Pseudomonas syringae (22).
However, the details surrounding the specific role of Lon in
bacterial virulence are not clearly defined. A comprehensive
understanding of the kinetic mechanism of Lon protease
could prove useful in the future for targeting specific
homologues with inhibitors. Because the protease and
ATPase activity of Lon are inevitably intertwined, a clear
understanding of the role of ATP hydrolysis activity in the
enzyme is necessary.
The broadly defined in vivo function of Lon includes
degrading misfolded or damaged proteins as well as shortlived regulatory proteins (4, 7, 9). Few physiological
substrates of Lon have been identified because the substrate
specificity is not well understood. One known substrate of
the Escherichia coli Lon homologue is the λN protein (7,
23). We have previously developed a small fluorescent
peptide mimic of the λ N protein containing a single cleavage
site (S3;1 comprised of 10% fluorescent S1 peptide and 90%
S2, the nonfluorescent analogue of S1) in order to easily
monitor the kinetics of the E. coli Lon system (24, 25).
Although it is known that ATP mediates the protease activity
in Lon, little definitive evidence exists for the timing of
events and kinetic coordination of the two activities. Lon is
known to bind to ADP with higher affinity than ATP, and
the ATPase as well as peptidase activities of Lon are
inhibited by ADP (25, 26). Nonhydrolyzable analogues of
ATP such as AMPPNP, which do not generate ADP, also
10.1021/bi060809+ CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/23/2006
Reproduced with permission from D. Vineyard et al., Biochemistry 45, 11432 (2006).
Copyright 2006 American Chemical Society.
224
Enzyme Form in Peptide-Stimulated ATPase Mechanism
Biochemistry, Vol. 45, No. 38, 2006 11433
support Lon-mediated peptide cleavage with reduced catalytic
efficiency (24). Although many observations have been
made, it is not clear precisely how ADP release contributes
to the catalytic mechanism of Lon. Using pre-steady-state
kinetic methods, we have determined that there is a burst of
ATP hydrolysis activity prior to the turnover of peptidase
activity (27). The burst activity indicates that a step following
ATP hydrolysis is rate limiting. This would be consistent
with ADP release being rate limiting. The pre-steady-state
ATPase activity was unusual because it displayed half-site
reactivity, presumably due to the existence of two ATPase
sites in Lon [Kd,high affinity ) 0.52 µM (26, 27), Kd,low affinity )
10 µM (26)]. The two ATPase sites are a result of monomeric
subunits having different ATPase behavior in the oligomeric
form of Lon, but they could not be distinguished structurally
because only one identical sequence for an ATPase motif
exists in each monomeric subunit. Therefore, kinetic studies
to further investigate the differences in the activities of the
high- and low-affinity ATPase sites in Lon protease were
performed (28). The experiments presented in this referenced
study capitalized on the differing affinities of the two sites.
By manipulating the concentration of ATP present, the
activity of the high- and low-affinity sites could be monitored
separately. The results showed that at stoichiometric concentrations of Lon and ATP (6 µM Lon, 6 µM ATP) the
high-affinity sites were slowly processing ATP at 0.01 s-1.
Although a previously published pulse-chase experiment
already confirmed that only one set of sites was responsible
for the pre-steady-state burst (27), we examined the lowaffinity ATPase sites by blocking the high-affinity ATPase
sites with stoichiometric amounts of unlabeled nucleotide.
The low-affinity sites hydrolyzed ATP faster (17 s-1) than
the high-affinity sites and were found to be solely responsible
for the pre-steady-state burst in ATPase activity. The activity
of each site apparently had no effect on the other’s activity.
Typically, a 2-5-fold stimulation of steady-state ATPase
activity is observed in the presence of peptide or protein
substrate (5, 25, 29). Interestingly, this stimulation was lost
if the high-affinity ATPase sites were blocked with stoichiometric amounts of ATP, suggesting that the peptide substrate
stimulates steady-state ATP hydrolysis at the high-affinity
sites. However, given the information gleaned from the
present study, the high-affinity sites seem to essentially be
noncatalytic. So the reduced steady-state activity noted in
the past study was more likely a result of ADP product
inhibition of the low-affinity sites, rather than the peptide
1 Abbreviations: AMPPNP, adenylyl 5-imidodiphosphate; DTT,
dithiothreitol; Abz, anthranilamide; Bz, benzoic acid amide; HBTU,
O-benzotriazole N,N,N′,N′-tetramethyluronium hexafluorophosphate;
HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; Tris,
2-amino-2-(hydroxymethyl)-1,3-propanediol; KPi, potassium phosphate;
Mg(OAc)2, magnesium acetate; KOAc, potassium acetate; SBTI,
soybean trypsin inhibitor; PEI-cellulose, polyethylenimine-cellulose;
PBP, phosphate binding protein; MDCC, 7-diethylamino-3-[[[(2maleimidyl)ethyl]amino]carbonyl]coumarin; MDCC-PBP, A197C mutant of PBP labeled with MDCC; MANT, 2′- (or 3′-) O-(Nmethylanthraniloyl); PNPase, purine nucleoside phosphorylase; MEG,
7-methylguanosine; Pi, inorganic phosphate; S2, a nonfluorescent
analogue of S3 that is degraded by Lon identically as S3 and is used
in the ATPase reactions to conserve the fluorescent peptide (S3)
YRGITCSGRQK(Bz); S3, a mixed peptide substrate containing 10%
of the fluorescent peptide Y(NO2)RGITCSGRQK(Abz) and 90% S2;
NTP, nucleotide triphosphate; SSD, the substrate sensor and discriminatory domain in Lon which is thought to interact with peptide substrate,
resulting in allosteric behavior.
225
not being able to stimulate activity of the low-affinity ATPase
sites.
These previous pre-steady-state kinetic studies of E. coli
Lon protease have only addressed the hydrolysis portions
of the ATPase mechanism at the high- and low-affinity sites
(27, 28). In the present study the rate of ATP binding and
ADP release from the two ATPase sites was investigated in
order to achieve a better understanding of the overall kinetic
mechanism. We used the fluorescent 2′- (or 3′-) O-(Nmethylanthraniloyl) (MANT) nucleotide analogues to accomplish this goal. In order to use this analogue, MANTATP was first shown to support S3 peptide hydrolysis, induce
a conformational change, and be hydrolyzed by E. coli Lon
protease comparably to ATP. The on rate of binding to the
high- and low-affinity ATPase sites was measured and found
to be on the order of 105 M-1 s-1. A conformational change
upon nucleotide binding was also detected, confirming
previous hypotheses (30). The off rate of ADP, however,
differed slightly for each of the ATPase sites, and although
this step was proposed to be rate limiting in steady-state
studies, ADP release only limited the low-affinity ATPase
turnover. Because S2 peptide affected none of the pre-steadystate rate constants, we propose a novel enzyme form that
exists after the first round of cleavage which undergoes
catalytic ATPase turnover in the presence of peptide. We
therefore revise our current kinetic model to accommodate
the newly obtained results.
MATERIALS AND METHODS
Materials. Nucleotides were purchased from Sigma or ICN
Biomedical. PNPase and 7-MEG were purchased from
Sigma. Fmoc-protected amino acids, Boc-Abz, Fmocprotected Lys Wang resin, and HBTU were purchased from
Advanced ChemTech and Nova Biochem. Tris, HEPES,
SBTI, and TPCK-treated trypsin were purchased from Fisher.
MANT-AMPPNP and MDCC were purchased from Molecular Probes. Cloning reagents were purchased from Promega,
New England BioLabs Inc., Invitrogen, and USB Corporations. Oligonucleotides were purchased from Integrated DNA
Technologies Inc.
General Methods. Peptide synthesis and protein purification procedures were performed as described previously (24).
Synthesis of MANT-ATP and MANT-ADP was performed
as described previously (31, 32). All enzyme concentrations
were reported as Lon monomer concentrations. All reagents
are reported as final concentrations. Unless otherwise stated
all experiments were performed at 37 °C.
Cloning and Purification of Phosphate Binding Protein
(PBP). The phosphate binding protein (PBP) gene with the
attached phoS signal sequence was amplified from genomic
DNA of the DH5R strain of E. coli using the forward primer
5-GGAATTCCATATGAAAGTTATGCGTACC-3′ and the
reverse primer 5′-CCCAAGCTTTTATTAGTACAGCGG3′. An A197C mutation was then introduced using PCR
primer site-directed mutagenesis and the additional forward
primer 5′-GTTGAATATTGTTACGCGAAG-3′ and reverse
primer 5′-CCTCGCGTAACAATATTCAAC-3′. The resulting product was cloned into the HindIII and NdeI sites of
the pET-24c(+) vector and the resulting plasmid named
pHF019. This subcloned phosphate binding protein from E.
coli DH5R contained one residue (Y306F) which differed
11434 Biochemistry, Vol. 45, No. 38, 2006
Vineyard et al.
from the originally cloned protein. pHF019 was overexpressed in BL21(DE3), selected for with 30 µg/mL kanamycin, and induced at OD600 ) 1.5 with 1 mM IPTG. The
protein was isolated by osmotic lysis and purified to
homogeneity as reported previously by Martin R. Webb and
colleagues (National Institute of Medical Research, Mill Hill,
London). This purification procedure and labeling of C197
with MDCC are described in detail in ref 33. The purified
labeled protein exhibits activity (excitation 425 nm, emission
465 nm) that is comparable to that observed in the PBP
purified from the original cell strain (a generous gift from
Susan Gilbert, University of Pittsburgh) developed by Webb
and colleagues.
Steady-State MANT-ATPase Assays Using MDCC-PBP.
Steady-state velocity data for MANT-ATP and ATP were
measured using an assay to detect inorganic phosphate (Pi)
release as described previously (33, 34). Reactions contained
50 mM Tris at pH 8.0, 5 mM Mg(OAc)2, 2 mM DTT, 150
µM 7-methylguanosine (MEG), 0.05 unit/mL PNPase, 300
or 150 nM E. coli Lon, (500 µM S2 peptide, and 25 µM-1
mM MANT-ATP or ATP. ATPase activity was monitored
on a Fluoromax 3 spectrofluorometer (Horiba Group) where
fluorescent MDCC-PBP was excited at 425 nm and emitted
at 465 nm. The velocity reactions were equilibrated at 37
°C for 1 min and initiated with the addition of Lon. Initial
velocities were determined from plots of the amount of Pi
released versus time. All assays were performed at least in
triplicate, and the kinetic parameters were determined by
fitting the averaged rate constant data with eq 1 using the
nonlinear regression program KaleidaGraph (Synergy) version 3.6:
Tryptic Digestions. Tryptic digest reactions were monitored
as described previously (28, 30). Briefly, 1.4 µM Lon in a
reaction mixture containing 50 mM HEPES (pH 8.0), 5 mM
Mg(OAc)2, 2 mM DTT, (800 µM S2 peptide, and 1 mM
ATP, 1 mM MANT-ATP, 1 mM ADP, 1 mM MANT-ADP,
1 mM AMPPNP, or 1 mM MANT-AMPPNP was started
by the addition of 1/50 (w/w) TPCK- (N-p-tosyl-L-phenylalanine chloromethyl ketone) treated trypsin with respect
to Lon. At 0 and 30 min, a 3 µL reaction aliquot was
quenched in 3 µg of soybean trypsin inhibitor (SBTI)
followed by boiling. The quenched reactions were resolved
by 12.5% SDS-PAGE analysis and visualized with Coomassie brilliant blue.
MANT-ATP Binding Time Courses by Fluorescent Stopped
Flow. Pre-steady-state experiments were performed on a
KinTek Stopped Flow controlled by the data collection
software Stop Flow version 7.50 β. The sample syringes were
maintained at 37 °C by a circulating water bath. Syringe A
contained 5 µM E. coli Lon monomer with and without 500
µM S2 peptide, 5 mM Mg(OAc)2, 50 mM HEPES, pH 8,
75 mM KOAc, and 5 mM DTT. Syringe B contained varying
amounts of MANT-ATP, MANT-dATP, MANT-ADP, or
MANT-AMPPNP (1-100 µM), 5 mM Mg(OAc)2, 50 mM
HEPES, pH 8, 75 mM KOAc, and 5 mM DTT. MANTnucleotide binding was detected by an increase in fluorescence (excitation 360 nm, emission 450 nm) resulting from
rapid mixing of the syringe contents in the sample cell. The
resulting exponential data were a result of averaging at least
four traces. All experiments were performed at least in
triplicate. The averaged time courses were fit with the
equation:
kobs ) kcat[ATP]/(Km + [ATP])
Y ) (A1 exp-k1t + C1) + (A2 exp-k2t + C2)
(1)
where kobs is the observed rate constant in s-1, kcat is the
maximal rate in s-1, [ATP] is the nucleotide concentration
in µM, and Km is the Michaelis-Menten constant in µM.
Peptidase Methods. Peptidase activity was monitored on
a Fluoromax 3 spectrofluorometer (Horiba Group) as described previously (25). Assays contained 50 mM HEPES,
pH 8.0, 75 mM KOAc, 5 mM DTT, 5 mM Mg(OAc)2, 200
nM E. coli Lon, 150 µM MANT-ATP or ATP, 50-150 µM
S1 peptide (100% fluorescent), and 200 µM-1.5 mM S3
peptide (10% fluorescent S1 peptide, 90% nonfluorescent
analogue S2 peptide) (excitation 320 nm, emission 420 nm).
Initial velocities were determined from plots of relative
fluorescence versus time. All assays were performed at least
in triplicate, and the kinetic parameters were determined by
fitting the averaged rate constant data with eq 2 using the
nonlinear regression program KaleidaGraph (Synergy) version 3.6:
k ) kmax[S]n/(K′ + [S]n)
(2)
where k is the observed rate constant being measured in units
of seconds, kmax is the maximum rate constant referred to as
kss,S3 in units of s-1, [S] is the variable peptide substrate in
units of µM, K′ is the Michaelis constant for [S], and n is
the Hill coefficient. The Ks (µM) is calculated from the
relationship log K′ ) n log Ks, where Ks is the [S] required
to obtain 50% of the maximal rate constant of the reaction
referred to as Km,ATP or Km,MANT-ATP.
226
(3)
where t is time in seconds, A1 and A2 are amplitudes for the
first and second exponential phases, respectively, in relative
fluorescence units, k1 and k2 are the observed rate constants
for the first and second exponential phases in seconds, and
C1 and C2 are constants.
MANT-ADP Release Time Courses by Fluorescent Stopped
Flow. Pre-steady-state experiments were performed on a
KinTek Stopped Flow controlled by the data collection
software Stop Flow version 7.50 β. The sample syringes were
maintained at 37 °C by a circulating water bath. Syringe A
contained 5 µM E. coli Lon monomer with and without 500
µM S2 peptide which was preincubated with varying
amounts of MANT-ADP (10 min), MANT-ATP, and MANTdATP (30 min, 37 °C) (0.05-200 M), in 5 mM Mg(OAc)2,
50 mM HEPES, pH 8, 75 mM KOAc, and 5 mM DTT.
Syringe B contained 1 mM ADP, 5 mM Mg(OAc)2, 50 mM
HEPES pH 8, 75 mM KOAc, and 5 mM DTT. MANT-ADP
release was detected by a decrease in fluorescence (excitation
360 nm, emission 450 nm) resulting from rapid mixing of
syringe contents in the sample cell. The resulting exponential
data were a result of averaging at least four traces. All
experiments were performed in triplicate and the averaged
time courses fit with eq 3.
Double mixing experiments were performed as above with
the following exceptions. The valve for syringe C was opened
on the KinTek Stopped Flow and the delay line calibrated
at 33 µL, resulting in a second push volume of 46 µL.
Syringe A contained 5 mM E. coli Lon monomer and 5 mM
Enzyme Form in Peptide-Stimulated ATPase Mechanism
Biochemistry, Vol. 45, No. 38, 2006 11435
ADP in 5 mM Mg(OAc)2, 50 mM HEPES, pH 8, 75 mM
KOAc, and 5 mM DTT. Syringe B contained 1 mM ADP
with or without 500 µM S2 in 5 mM Mg(OAc)2, 50 mM
HEPES, pH 8, 75 mM KOAc, and 5 mM DTT. Syringe C
contained 100 µM MANT-ADP or ATP in 5 mM Mg(OAc)2,
50 mM HEPES, pH 8, 75 mM KOAc, and 5 mM DTT. The
contents of syringes A and C were rapidly mixed with a first
reaction time of 50 s for MANT-ATP and 3 ms for MANTADP. The second push would then mix the developed
reaction from syringes A and C with the contents of syringe
B in the observation cell in order to monitor the release of
MANT-ADP from Lon over 30 s. All experiments were
performed at least in triplicate and the averaged time courses
fit with a single exponential equation.
RESULTS
Steady-State Characterization of the MANT-ATPase ActiVity of Lon. In order to perform pre-steady-state stopped-flow
experiments to determine the rate constants associated with
ATP binding and ADP release, a fluorescent analogue of
ATP was needed. To determine if the N-methylanthraniloyl
(MANT) fluorescently labeled ATP was also a substrate of
Lon protease, and thus an appropriate analogue of unlabeled
ATP, the steady-state kinetics of MANT-ATP hydrolysis
were examined in comparison to ATP hydrolysis. This was
accomplished using a coupled assay system with MDCCPBP on a Fluoromax 3 spectrofluorometer (Horiba Group)
as described previously (33, 34). Phosphate binding protein
(PBP) is the product of the phoS gene in E. coli which is
induced when the levels of Pi are low, is localized to the
periplasmic space, and is implicated in the transport of Pi
(33, 35). Martin Webb and colleagues (National Institute for
Medical Research) introduced the mutation A197C in PBP
in order to covalently label the introduced cysteine with a
fluorophore (33). By doing this, they created a probe for Pi
which can rapidly measure micromolar concentrations released from enzymes in real time due to the increase in
fluorescence resulting from MDCC-PBP binding Pi. In order
to reduce the extraneous Pi contamination, the cuvettes were
soaked in a phosphate “mop” solution consisting of 150 µM
MEG and 0.05 unit/mL PNPase, which removes excess
phosphate by converting it to ribose 1-phosphate as described
previously (33, 36, 37). At these concentrations there was
no competition between MDCC-PBP and the phosphate mop
system for Pi. The MDCC fluorophore was excited at 425
nm, and fluorescence emission was detected at 465 nm. The
relative fluorescence generated from MDCC-PBP was correlated to the concentration of Pi by a calibration curve where
known concentrations of Pi were linearly increased and
plotted versus the resulting change in fluorescent signal (data
not shown). The observed rate constants were measured at
varying concentrations of nucleotide from the linear region
of initial velocity plots of Pi released over time. When plotted
versus the concentration of nucleotide, both ATP and
MANT-ATP yielded Michaelis-Menten kinetics as shown
in Figure 1. This kcat/Km profile was performed in the absence
and presence of S2 peptide for both ATP and MANT-ATP.
The steady-state kinetic parameters are summarized in Table
1. Since the kcat/Km values are similar for MANT-ATP and
ATP, we conclude that Lon hydrolyzes the fluorescent
analogue in a comparable manner to ATP.
227
FIGURE 1: Steady-state kinetics of ATP and MANT-ATP hydrolysis
by E. coli Lon. ATPase activity was monitored using a coupled
assay system where MDCC-PBP binds the Pi released from the
hydrolysis of ATP and MANT-ATP by Lon, resulting in an increase
in fluorescence over time. The initial rates obtained from the time
courses were converted to kobs values by dividing the steady-state
rates of the reactions by [Lon]. The ([) and (2) represent intrinsic
and S2-stimulated ATPase activity, respectively, at varying concentrations of ATP. The (b) and (9) represent intrinsic and S2stimulated MANT-ATPase activity, respectively, at varying concentrations of MANT-ATP. The data are reported as the average
values of at least three trials and were fit with eq 1 to obtain the
kinetic parameters kcat and Km. The kinetic parameters for intrinsic
and S2-stimulated ATPase activity were kcat ) 0.34 ( 0.01 s-1,
Km ) 18 ( 4 µM ([) and kcat ) 1.1 ( 0.1 s-1, Km ) 31 ( 8 µM
(2), respectively. The kinetic parameters for intrinsic and S2stimulated MANT-ATPase activity were kcat ) 0.53 ( 0.02 s-1,
Km ) 43 ( 5 µM (b) and kcat ) 1.1 ( 0.1 s-1, Km ) 37 ( 6 µM
(9), respectively. The rate constants are also summarized in Table
1.
Steady-State Analysis of MANT-ATP-Dependent S3 CleaVage by Lon. In addition to demonstrating that MANT-ATP
is hydrolyzed by Lon protease, it was necessary to ensure
that MANT-ATP was also capable of supporting S3 peptide
cleavage in a comparable manner to ATP. The fluorescent
peptidase assay previously employed to monitor the kinetics
of ATP-mediated S3 cleavage (25, 27, 30) was performed
to compare the ability of MANT-ATP to support S3 cleavage
compared to ATP. The observed steady-state rate constants
of S3 cleavage (kss,S3) were determined at varying concentrations of S3 (50-1500 µM) and 150 µM MANT-ATP or
ATP. Because the predominant enzyme form under these
conditions is Lon-ATP, the observed rate constants are a
reflection of the effect of nucleotide hydrolysis rather than
binding. The S3 hydrolysis reactions were monitored by the
increase of fluorescence over time, which when calibrated
reflects the amount of peptide hydrolyzed over time. The
observed steady-state rate constants were then plotted as a
function of S3 concentration, and this yielded a sigmoidal
plot as shown in Figure 2. The kinetic parameters obtained
from fitting the data in Figure 2 with the Hill equation are
summarized in Table 2. Although the value of n determined
here is slightly higher than that determined previously (n )
1.6), both are approximately equal to 2, and the discrepancy
11436 Biochemistry, Vol. 45, No. 38, 2006
Vineyard et al.
Table 1: MANT-ATP and ATP Steady-State Kinetic Parameters Associated with ATP Hydrolysis
kcat (s-1)
Km (µM)
kcat/Km (103 M-1 s-1)
intrinsic
ATPase
intrinsic
MANT-ATPase
S2-stimulated
ATPase
S2-stimulated
MANT-ATPase
0.34 ( 0.01
18 ( 4
19
0.53 ( 0.02
43 ( 5
12
1.1 ( 0.1
31 ( 8
35
1.1 ( 0.1
37 ( 6
30
FIGURE 2: Steady-state kinetics of ATP- and MANT-ATPdependent S3 cleavage by E. coli Lon. 150 µM ATP and MANTATP [5 mM Mg(OAc)2] dependent peptidase activity was monitored
using a modified FRET assay system where the cleavage of the S3
peptide containing a fluorescent donor and quencher results in
donor/quencher separation and an increase in fluorescence over
time. The initial steady-state rates of S3 cleavage were obtained
from the time courses of peptide cleavage at varying [S3] and
converted to steady-state rate constants kss,S3 by dividing by the
[Lon]. Both ATP (b) and MANT-ATP (9) mediated S3 cleavage
yielded sigmoidal curves which were fit with eq 2. The resulting
kinetic parameters for ATP-mediated S3 cleavage (b) were kss,S3
) 9.6 ( 0.2 s-1, Km,ATP ) 240 ( 9 µM, and n ) 2.4 ( 0.1 while
the parameters for MANT-ATP-mediated S3 cleavage were kss,S3
) 5.9 ( 0.3 s-1, Km,MANT-ATP ) 360 ( 30 µM, and n ) 2.5 ( 0.4.
The rate constants are also summarized in Table 2.
Table 2: MANT-ATP and ATP Steady-State Kinetic Parameters
Associated with S3 Cleavage
ATP
MANT-ATP
kss,S3 (s-1)
Km (µM)
n
9.6 ( 0.2
5.9 ( 0.3
240 ( 9
360 ( 30
2.4 ( 0.2
2.5 ( 0.4
is most likely due to the slightly differing experimental
methods. Because the steady-state kinetic parameters were
comparable to one another (Table 2), MANT-ATP supports
S3 cleavage in a similar manner as ATP. In addition, these
rate constants were comparable to the previously published
kcat and Km values for ATP-mediated S3 cleavage (25, 30).
So although the nucleotide MANT-ATP contains a fluorescent label, it still supports S3 degradation in a similar manner
to ATP and therefore is acceptable to use as an ATP
analogue.
Limited Tryptic Digestion Probes the ATP-Dependent
Conformational Change in Lon. Previously (30), we have
utilized limited tryptic digestion to probe the functional role
of nucleotide binding to Lon. This revealed an adeninespecific conformational change associated with nucleotide
binding, which can primarily be monitored by the stability
of a 67 kDa fragment of Lon. When sequenced, this fragment
was composed of the ATPase, the substrate sensor and
discriminatory domain (SSD), and protease domains of Lon.
To ensure that the introduction of the fluorophore on MANTATP does not affect the nucleotide’s ability to induce this
conformational change in Lon, we subjected 1.4 µM Lon to
limited tryptic digestion (1/50 w/w) in the presence of no
228
FIGURE 3: Limited tryptic digest shows that MANT-ATP and ATP
induce the same conformational change in E. coli Lon protease.
SDS-PAGE visualized by Coomassie brilliant blue show 1.4 µM
Lon digested with a limiting amount of trypsin and quenched with
SBTI at the indicated times as described in Materials and Methods.
Lane 1 shows the molecular markers in kilodaltons (from top to
bottom): 183, 114, 81, 64, 50, 37, 26, and 20. Lanes 2 and 3 contain
Lon without nucleotide, lanes 4 and 5 contain Lon + 1 mM ATP,
lanes 6 and 7 contain Lon + 1 mM MANT-ATP, lanes 8 and 9
contain Lon + 1 mM ADP, lanes 10 and 11 contain Lon + 1 mM
MANT-ADP, lanes 12 and 13 contain Lon + 1 mM AMPPNP,
and lanes 14 and 15 contain Lon + 1 mM MANT-AMPPNP.
nucleotide and saturating amounts (1 mM) of ATP, MANTATP, ADP, MANT-ADP, AMPPNP, and MANT-AMPPNP.
At 0 and 30 min the reaction was quenched with soybean
trypsin inhibitor (SBTI) and resolved on a 12.5% SDSPAGE as shown in Figure 3. This figure demonstrates that
MANT-ATP (lanes 4 and 5) induces the same conformational
change as ATP (lanes 2 and 3). MANT-ADP (lanes 10 and
11) and MANT-AMPPNP (lanes 14 and 15) also induce the
same conformational change as ADP (lanes 8 and 9) and
AMPPNP (lanes 12 and 13) ,respectively. S2 peptide did
not change the digestion pattern (data not shown), indicating
that peptide does not induce any conformational change that
is detectable by tryptic digestion.
Determining the Rate of MANT-ATP Binding Using
Fluorescent Stopped Flow. Since MANT-ATP is hydrolyzed,
supports peptide cleavage, and induces the same conformational change as ATP, the MANT-nucleotide fluorescent
analogues can be used to study individual kinetic steps along
the Lon reaction pathway. The on rate of MANT-ATP
binding was monitored by stopped-flow fluorescence spectroscopy because an increase in fluorescence is detected upon
Lon binding to MANT-ATP. Rapidly mixing 5 µM Lon both
in the presence and in the absence of saturating amounts
(500 µM) of S2 peptide with amounts of MANT-ATP
varying from 1 µM to 100 µM resulted in time courses that
were best fit using a double exponential equation. A
representative time course is shown in Figure 4a with the
solid black line demonstrating the fit of the double exponential equation (Materials and Methods). During the synthesis of MANT-ATP, the MANT fluorophore attaches to
both the 2′- and 3′-hydroxyl on the ribose (32). The mixture
Enzyme Form in Peptide-Stimulated ATPase Mechanism
Biochemistry, Vol. 45, No. 38, 2006 11437
Table 3: Rate Constants Associated with Adenine Nucleotide
Binding As Determined by Fluorescent Stopped Flow
MANTnucleotide
ATP
ATP + S2
ADP
kon,MANT-Nu
(105 M-1 s-1)
koff, MANT-Nu
(s-1)
kon,2
(s-1)
6.8
6.8
6.9
11
10
9.5
0.46a (0.13)a
9.7
0.44a (0.13)a
22
6.6a (0.16)a
24
6.7a (0.16)a
4.1 ( 1.2
3.7 ( 1.2
3.9 ( 0.9
ADP + S2
6.1
AMPPNP
3.2
AMPPNP + S2
2.1
3.7 ( 0.9
6.7 ( 2.8
7.1 ( 4.7
a
Values determined by single mixing release rate experiments where
Lon was preincubated with MANT nucleotide and then rapidly mixed
with excess unlabeled nucleotide. These values are more accurate
because the off rates are being directly measured in the experiment.
Two off rates for the nucleotides are detected in this experiment. The
off rate from the high-affinity site is in parentheses, and the other
number represents the off rate from the low-affinity site.
FIGURE 4: MANT-ATP binding to Lon. (a) Representative time
course of MANT-ATP binding to Lon. Five micromolar Lon was
rapidly mixed with varying amounts of MANT-ATP (excitation
360 nm, emission 450 nm) in the presence and absence of 500 µM
S2 peptide, and the increase in fluorescence was monitored for 1
s. The time courses were fit with a double exponential (eq 3, solid
black line). The two resulting rate constants (kon,1, kon,2) are shown
in (b) at varying concentrations of MANT-ATP. (b) On-rate
constants of MANT-ATP binding plotted versus [MANT-ATP].
kon,1 shows a linear dependence on [MANT-ATP]. The on rate for
MANT-ATP is determined from the slope of the line (kon,MANT-ATP
) 6.8 × 105 M-1 s-1). The off rate for MANT-ATP can be
estimated from the y-intercept of the line (koff,MANT-ATP ) 11 s-1).
kon,2 remains constant at 5 s-1, likely indicating a conformational
change associated with nucleotide binding. These rate constants
are also summarized in Table 3.
of the two isomers could result in fluorescent changes which
are unrelated to the enzyme nucleotide interaction. In order
to ensure this was not the reason that biphasic time courses
were observed, control experiments with MANT-dATP were
performed which also resulted in biphasic time courses (data
shown in Supporting Information). Two observed rate
constants (kon,1, kon,2) are extracted from the double expo-
229
nential equation and plotted versus the concentration of
MANT-ATP as shown in Figure 4b. The rate constants
associated with MANT-ATP binding are identical in the
presence and absence of S2 peptide, indicating that peptide
has no effect on the binding of nucleotide (Table 3). The
data shown in Figure 4b are representative of MANT-ATP
binding in the absence of S2. The observed rate constants,
kon,1, are linearly dependent on the concentration of MANTATP (Figure 4b). The slope of the line, 6.8 × 105 M-1 s-1,
yields the on rate of MANT-ATP binding, and the y-intercept
is an estimate of the off rate (11 s-1). Furthermore when the
Kd is calculated by dividing the off rate by the on rate, the
value (16 µM) is similar to the previously published value
of 10 µM (26) for the low-affinity ATPase site (28). The
range of nucleotide concentration needed to probe the highaffinity site (0.05-5 µM) was beyond the limit of detection
at the lower concentrations and indistinguishable from the
low-affinity site at the higher concentrations. The ATP off
rate for the high-affinity ATPase site could not be detected
using this method for the same reason. Instead, MANTAMPPNP, a nonhydrolyzable analogue, was used as elaborated on in the Discussion. The observed rate constant, kon,2
∼ 5 s-1 (Figure 4b, Table 3), shows no dependence on the
concentration of MANT-ATP. The lack of dependence on
nucleotide concentration could be indicative of a conformational change step associated with MANT-ATP binding. This
is supported by the tryptic digest which also suggests a
conformational change upon nucleotide binding which
protects Lon from degradation by trypsin (Figure 3) (30).
The same binding experiment was conducted to determine
the rate of MANT-ADP and MANT-AMPPNP binding to
Lon, and the rate constants were comparable to MANT-ATP.
The rate constant, kon,2, did not vary with the concentration
of nucleotide, which is again consistent with a conformational
change (Table 3).
Determining the Rate of MANT-ADP Release Using
Fluorescent Stopped Flow. The rate constant associated with
MANT-ADP release could also be monitored using fluorescence stopped-flow spectroscopy because there is a decrease
in fluorescence upon the dissociation of MANT-ADP from
Lon. Five micromolar Lon in the presence and absence of
500 µM S2 peptide was preincubated with 0.05-200 µM
11438 Biochemistry, Vol. 45, No. 38, 2006
Vineyard et al.
MANT-ADP and rapidly mixed with excess unlabeled (1
mM) ADP. This resulted in a decrease in fluorescence which
was best fit with a double exponential (Materials and
Methods). A representative time course is shown in Figure
5a, where the solid black line represents the fit of the double
exponential equation. To ensure that the two phases were
not a result of esterification of the fluorophore on MANTATP, control experiments were performed using MANTdADP which were also biphasic (data shown in Supporting
Information). The two rate constants (koff,1, koff,2) obtained
from the double exponential fit of the data are summarized
in Table 4. As one would expect, there is no dependence on
the concentration of MANT-ADP with either rate constant
because the release step being monitored is a unimolecular
event. The presence of S2 peptide also does not affect the
rate constants associated with MANT-ADP release regardless
of the order of addition. The average value for koff,1 was 0.46
( 0.03 s-1 and 0.14 ( 0.01 s-1 for koff,2. To ensure that the
MANT fluorescent label was not affecting the off rate, the
experiment was also performed where Lon was preincubated
with varying amounts of unlabeled ADP and rapidly mixed
with MANT-ADP, resulting in an increase in fluorescence.
The rate constants obtained from performing the experiment
in this way were identical (data not shown), assuring that
the fluorescent label was not affecting the observed rate
constants. MANT-ADP release was also monitored by
preincubating Lon with varying amounts of MANT-ATP for
30 min at 37 °C so all the MANT-ATP was hydrolyzed to
MANT-ADP. The resulting mixture was then rapidly mixed
with excess (1 mM) unlabeled ADP on the stopped flow.
These resulting time courses were again identical to those
where MANT-ADP was used directly (Table 4). This
indicates that if by performing the experiments in this way
we are indeed probing a pre- versus postcatalytic form of
Lon, the two forms do not release MANT-ADP at differing
rates. However, because there is not a clear mechanistic
understanding of how Lon turns over, the possibility that
we are isolating identical enzyme forms in the two experiments cannot be excluded.
The single mixing stopped-flow experiments discussed
above are set up so that every fluorescent molecule bound
to the enzyme is chased off. Because of the sensitivity of
the stopped-flow method even at low concentrations of
MANT-ADP the release rate from both sites was being
detected (Table 4). Therefore, stopped-flow double mixing
experiments were employed to uncouple MANT-ADP release
from the high- and low-affinity ATPase sites. These experiments allowed for the mixing of two of the reaction
components for a designated period of time prior to the
introduction of the third component and subsequent monitoring of the fluorescent signal. We demonstrated in a previous
publication that the high-affinity ATPase sites could be
blocked by preincubating 6 µM Lon with stoichiometric
amounts of ADP (28). Whether the ADP was generated in
situ or directly added in the preincubation with Lon, the presteady-state ATP hydrolysis activity at the low-affinity sites
remained unaffected. We utilized the high-affinity ATPase
site blocking technique in this study to isolate MANT-ADP
release from only the low-affinity site. To this end, 5 µM
Lon preincubated with 5 µM ADP (saturates high-affinity
sites) was rapidly mixed with 100 µM MANT-ADP (saturates low-affinity sites) for 3 ms. This reaction was subse-
230
FIGURE 5: MANT-ADP release from Lon. (a) Representative time
course of MANT-ADP release in single mixing experiments. Five
micromolar Lon was preincubated with varying amounts of MANTADP or MANT-ATP (excitation 360 nm, emission 450 nm) in the
presence and absence of 500 µM S2 peptide and rapidly mixed
with 1 mM ADP. The resulting decrease in fluorescence was
monitored for 30 s, and the time courses were fit with a double
exponential (eq 3, solid black line). Two averaged rate constants,
koff,1 ) 0.46 ( 0.03 s-1 and koff,2 ) 0.14 ( 0.01 s-1, resulted and
are summarized in Table 4. (b) Representative time course of ADP
release in double mixing experiments. The high-affinity ATPase
sites in 5 µM Lon were blocked with 5 µM ADP, and this mixture
was rapidly mixed with 100 µM MANT-ADP or MANT-ATP for
3 ms or 50 s, respectively. 1 mM ADP then mixes with the reaction
to displace the bound nucleotide, and the decrease in fluorescence
is monitored for 30 s. The time courses were fit with a single
exponential equation (solid black line), and the resulting rate
constant (koff,1 ) 0.5 ( 0.1 s-1) describes the release of ADP from
only the low-affinity sites.
quently mixed with 1 mM ADP ( 500 µM S2 and the
decrease in fluorescence monitored as in the above experiments. The same experiment was performed with 100 µM
Enzyme Form in Peptide-Stimulated ATPase Mechanism
Biochemistry, Vol. 45, No. 38, 2006 11439
Table 4: Summary of MANT-ADP Release Rate Constants from Single Mixing Stopped-Flow Experiments
intrinsic koff,1 (s-1)
S2-stimulated koff,1 (s-1)
intrinsic koff,2 (s-1)
S2-stimulated koff,2 (s-1)
[MANT-Nu] (µM)
MANT-ADP
MANT-ATP
MANT-ADP
MANT-ATP
MANT-ADP
MANT-ATP
MANT-ADP
MANT-ATP
0.05
0.1
0.5
50
200
0.42 ( 0.01
0.52 ( 0.01
0.47 ( 0.02
0.42 ( 0.01
0.46 ( 0.06
0.43 ( 0.01
0.44 ( 0.01
0.51 ( 0.16
0.46 ( 0.04
NDa
0.45 ( 0.01
0.45 ( 0.01
0.47 ( 0.02
0.45 ( 0.01
0.40 ( 0.04
0.44 ( 0.01
0.46 ( 0.02
0.46 ( 0.02
0.49 ( 0.05
ND
0.44 ( 0.01
0.12 ( 0.01
0.13 ( 0.01
0.15 ( 0.01
0.12 ( 0.01
0.14 ( 0.01
0.12 ( 0.01
0.12 ( 0.01
0.13 ( 0.02
0.13 ( 0.02
0.12 ( 0.04
ND
0.12 ( 0.01
0.12 ( 0.01
0.13 ( 0.01
0.15 ( 0.01
0.12 ( 0.01
0.14 ( 0.01
0.12 ( 0.01
0.12 ( 0.01
0.14 ( 0.02
0.14 ( 0.02
0.13 ( 0.04
ND
0.12 ( 0.01
a
ND: these values were not determined.
MANT-ATP except the delay time was increased to 50 s to
allow for complete hydrolysis of MANT-ATP at the lowaffinity sites. Figure 5b illustrates a representative time course
from the double mixing experiments. The time course is
single exponential in nature presumably because MANTADP release from only the low-affinity sites is being
monitored. The solid black line shows the fit of a single
exponential equation resulting in a rate constant of 0.5 (
0.1 s-1 for ADP release at the low-affinity sites. By inference,
the second rate constant detected in the single mixing
experiments (koff,2 ) 0.14 s-1, Figure 5a, Table 4) must
describe the release of ADP from the high-affinity ATPase
site.
DISCUSSION
Lon is a homohexameric ATP-dependent protease in which
the ATPase and protease domains are located within each
enzyme subunit. Using MANT-nucleotides as fluorescent
probes, we were able to compare the kinetics of ATP,
AMPPNP, and ADP binding to and release from E. coli Lon.
The pre-steady-state binding experiments performed in this
study verified that the on rate of ATP binding is comparable
to AMPPNP and that a conformational change occurs after
nucleotide binding (Table 3). Therefore, the difference
between ATP- and AMPPNP-activated peptide cleavage
occurs after nucleotide binding. Although there is only one
ATP binding domain on each monomeric subunit in Lon,
studies have identified two binding affinities for ATP
(Kd,low affinity ) 10 µM, Kd,high affinity < 1 µM) (26). Additional
experiments performed in this laboratory isolated the binding
constant at the high-affinity site to be 0.52 ( 0.02 µM (28).
When the hydrolysis activity of each ATPase site was
examined using single turnover experiments, the high-affinity
sites were found to hydrolyze ATP very slowly (0.01 s-1)
(28). The ATP binding experiments performed in the present
study encompassed a range of ATP concentrations that
included both the high- and low-affinity sites. Both sites were
found to have a similar on rate which is relatively fast (Table
3). Therefore, because the dissociation constant, Kd, is
defined by koff/kon, the high-affinity ATPase site must have
a slower koff than the low-affinity site. The value 10 s-1
obtained from the y-intercept of Figure 4b (Table 3) yields
an estimate of the koff of ATP for the low-affinity site. This
method was not sensitive enough to distinguish a second
value for the high-affinity sites because the off rate is not
being directly measured in the experiment. Instead, MANTAMPPNP was used as a probe because it is a nonhydrolyzable analogue of ATP which supports S2 peptide cleavage
and displays identical binding kinetics to Lon as MANTATP. The off rate could then be directly monitored using
231
the same methodology used for detecting the koff for MANTADP. Two observed rate constants for MANT-AMPPNP
release resulted, which did not vary in the presence of S2
peptide (Table 3). When the observed off rates were divided
by the on rate, the two Kd values obtained for AMPPNP
were 0.3 and 9 µM. These values are in close agreement
with the Kd values of the high- and low-affinity ATPase sites
determined previously [0.5 µM (28), 10 µM (26)]. Therefore,
it is likely that the koff values of ATP are the same as
AMPPNP.
To characterize the kinetics of ADP interacting with Lon,
we utilized single mixing stopped-flow experiments to
determine the off rate of MANT-ADP, which resulted in two
observed rate constants (koff,1 ) 0.46 ( 0.03 s-1, koff,2 )
0.14 ( 0.01 s-1, Table 4). However, only one rate constant
associated with the binding of ADP which is dependent on
the concentration of nucleotide was detected (6.9 × 105 M-1
s-1, Table 3). The difference between the two observed ADP
off rates is modest so when the off rates are divided by the
on rate, it yields two Kd values that differ only by 3-fold.
Since the original article that investigated the binding of
nucleotides to Lon had a detection limit of 1 µM for ATP
binding (26), it is not surprising that a subtle difference in
the Kd for ADP was not detected in the nanomolar range.
Furthermore, when the double mixing experiment was
performed with MANT-ATP, if the delay time was not long
enough to allow for complete hydrolysis of MANT-ATP at
the low-affinity site (Figure 1, 0.5 s-1), the off rate of
MANT-ATP as well as MANT-ADP was detected. As the
high-affinity ATPase site hydrolyzes ATP much more slowly
[0.01 s-1 (28)] than the low-affinity site (Figure 1, 0.5 s-1),
it is likely that ADP release at the high-affinity site occurs
much later than at the low-affinity site.
Kinetic studies performed previously indicated that ADP
release was the rate-limiting step along the reaction pathway
of Lon protease (26, 27, 38). This conclusion was supported
by a proposed ATP/ADP exchange model which was based
on data that showed that Lon was proteolytically “inactive”
when bound to ADP, that protein substrate allosterically
interacted with Lon to promote ADP release which was the
rate-limiting step, and that Lon was only proteolytically
“active” when the bound ADP was exchanged to ATP (38).
The fact that nonhydrolyzable analogues such as AMPPNP,
which does not generate ADP, support peptide cleavage at
a lower rate than ATP negates this proposed model. The
kinetic mechanism has been further investigated recently
using pre-steady-state kinetic techniques to determine the
timing of events along the pathway. ATP hydrolysis was
found to occur prior to peptide cleavage at the low-affinity
site (25, 27). Because burst kinetics was detected in the pre-
11440 Biochemistry, Vol. 45, No. 38, 2006
Vineyard et al.
steady-state time course of ATP hydrolysis, the rate-limiting
step must occur after ATP hydrolysis, and ADP release
would be consistent with this. The rate-limiting step should
also have a rate constant similar to the overall kcat for ATP
hydrolysis. As shown in Figure 1, the kcat for S2-stimulated
ATP hydrolysis is 1.1 and 0.53 s-1 for intrinsic ATP
hydrolysis. We have previously suggested that the S2
stimulation of the steady-state ATPase activity is a result of
the high-affinity sites interacting with peptide (28). Because
both of the rate constants identified in this study for MANTADP release were slower than the S2-stimulated kcat for ATP
hydrolysis, we questioned the validity of this suggestion.
When the timing of all of the pre-steady-state experiments
was examined in entirety, the high-affinity sites seem to be
essentially catalytically inactive. Prior pulse-chase (27) and
single turnover (28) experiments demonstrate that the highaffinity ATPase site is capable of hydrolyzing ATP if given
enough time; however, the steady-state rate is never affected
by the high-affinity ATPase activity. Furthermore, although
Lon (5 µM) undergoes multiple rounds of peptide cleavage
at limiting (500 nM) ATP, 100 µM peptide is cleaved prior
to the half-life of the high-affinity ATPase site reaction (28).
For these reasons it appears that the ATPase pre-steady-state
burst activity as well as the steady-state activity can be
attributed to the low-affinity sites. However, a detailed
understanding of how the S2 peptide is interacting with Lon
to stimulate steady-state ATPase activity is still necessary.
The steady-state kcat values can thus be attributed to lowaffinity ATPase activity which is stimulated in the presence
of peptide. Because the off rate of MANT-ADP from the
low-affinity site (0.46 s-1, Table 4) approaches the turnover
number for intrinsic MANT-ATPase activity (0.53 s-1, Table
1), ADP release is likely the rate-limiting step in this
pathway. Prior experiments have been performed which
suggested that the presence of peptide or protein substrate
facilitated the release of ADP from Lon. Menon and
Goldberg demonstrated this in two ways. After incubation
with [3H]ADP to establish an equilibrium the addition of
casein to the reaction resulted in the recovery of lesser
amounts of [3H]ADP-Lon complex over time than in the
absence of casein (38). They also noted that basal ATP
hydrolysis was inhibited by a greater percentage in the
absence versus the presence of casein (38). ThomasWohlever and Lee confirmed these observations using
steady-state ADP inhibition analyses of ATP-dependent S3
cleavage by Lon protease. They measured Kis and Kii values
which reflected affinity of Lon for ADP in the presence of
very low and high levels of S3, respectively. The affinity
for ADP was weakened in the presence of higher levels of
S3 (Kii > Kis) presumably by binding allosterically to promote
ADP/ATP exchange (25). Collectively, these results would
suggest that the rate of ADP release is possibly increased in
the presence of peptide or protein substrate. However, the
results of the stopped-flow MANT-ADP release experiments
summarized in Table 4 demonstrate that there is no difference
in the off rate in the presence of peptide (Table 4) or protein
substrate (data not shown). The rate constant for ATP binding
and the conformational change detected upon nucleotide
binding were also unaffected by peptide or protein substrate
(Table 3). As no pre-steady-state rate constant identified thus
far has been affected by S2 peptide, we therefore conclude
that the obtained pre-steady-state rate constants describe the
232
Scheme
1a
a
Form I represents oligomeric free enzyme shown for simplicity as
a dimer (bottom white circle ) peptidase domain, middle white oval
) substrate senor and discriminatory domain (SSD), top left blue oval
) low-affinity ATPase, and top right red oval ) high-affinity ATPase).
Forms II-IV represent intrinsic steady-state ATPase activity at the lowaffinity sites. Form V represents the ability of the high-affinity site to
hydrolyze ATP.
first round of ATP hydrolysis which is independent of
peptide. If peptide is present, it acts on a enzyme form
following the first round of ATP hydrolysis which is then
catalytically active and described by the peptide stimulated
kcat (1 s-1, Table 1). Further experimentation will have to be
performed to isolate the intermediates in this pathway.
The best estimate of a turnover number for the highaffinity sites is 0.01 s-1, as demonstrated using single
turnover methods described previously (28). However, the
high-affinity ATPase sites release ADP at 0.14 s-1, which
is faster than the turnover number (0.01 s-1). Thus another
step must be limiting turnover at the high-affinity site. Until
the activities at the two sites can be more easily uncoupled
this step will likely remain unknown. However, because
hydrolysis is so slow and appears to not contribute to the
ATP-dependent peptidase activity of Lon, it would not be
surprising if chemistry or a step prior to chemistry was rate
limiting for the high-affinity site.
An additional step along the pathway that we attempted
to monitor was the release of phosphate (Pi) from Lon. The
MDCC-PBP fluorescent coupled assay system used to
monitor the steady-state hydrolysis of MANT-ATP is the
ideal way to approach this. Unfortunately, the production of
Pi in the pre-steady-state was below the detection limit of
the MDCC-PBP method under our assay conditions. Phosphate release has been proposed to be negligible because
increasing amounts of phosphate do not inhibit the activity
of Lon (25) and Pi was never found to remain bound to Lon
as ADP was in equilibrium binding experiments (26, 38).
In summary, we have utilized pre-steady-state techniques
to determine the kinetic mechanism of the ATPase activities
of Lon. Collectively, our results allow us to refine the
previously constructed kinetic mechanism of E. coli Lon (25,
27). The revised model is illustrated in Scheme 1 in the
Enzyme Form in Peptide-Stimulated ATPase Mechanism
Scheme
Biochemistry, Vol. 45, No. 38, 2006 11441
2a
Scheme
3a
a E and F are different catalytic forms of Lon along the reaction
pathway.
Table 5: Comparison of Experimentally Obtained Rate Constants
with Those Determined from the Collective Fit of the Data to the
Kinetic Mechanism in Scheme 3
a As in Scheme 1, in form IV Lon has hydrolyzed the first round of
ATP at the low-affinity sites. Following ADP release from the lowaffinity ATPase site after the first turnover, if peptide substrate is
present, we propose enzyme forms VI and VII.
absence of peptide, and then Scheme 2 proposes a kinetic
model for ATPase activity in the presence of peptide.
Although Lon exists as a higher order oligomer shown in
partial crystal structures as a hexamer (15, 16, 39), we
represent it as a dimer in order to simplify the schemes. Each
Lon monomer contains four defined domains, the amino
terminus, ATPase, substrate sensor and discriminatory (SSD),
and the protease domain. The amino terminus is not
represented in the schemes for simplicity, the low-affinity
ATPase domains are represented as blue ovals, the highaffinity ATPase domains are represented as red ovals, the
SSD domain is represented as white ovals, and the peptidase
domain is represented as white circles. In Scheme 1, Lon in
its free enzyme (form I) binds to ATP at both the high- and
low-affinity ATPase sites at a fast rate (Table 3, kon,ATP )
6.8 × 105 M-1 s-1). The binding of nucleotide induces a
conformational change (form II) (25) measured in the
stopped-flow binding experiments at approximately 5 s-1
(Table 3). ATP is subsequently hydrolyzed at the low-affinity
ATPase sites at 11 s-1 (form IV) (27, 28), and ADP is
released from the low-affinity ATPase sites at 0.5 s-1 (Table
4). Turnover at the low-affinity ATPase sites then occurs in
the absence of peptide (forms II-IV) (Table 1, kcat ) 0.5
s-1) and is limited by ADP release. In a separate pathway
(forms IV and V) the high-affinity ATPase sites can
hydrolyze ATP at 0.01 s-1 (28) and release ADP at 0.1 s-1
(Table 4).
Scheme 2 represents the proposed kinetic mechanism for
ATPase activity in the presence of peptide. On the basis that
peptide affects only the steady-state ATPase activity of the
low-affinity sites, we propose that Lon adopts a different
form after its first turnover as an ATP-dependent protease,
i.e., from IV to VI. Enzyme form VI is speculative, although
previous tryptic digest studies tentatively support its existence
(28). Catalytic steady-state turnover can then occur (forms
VI-VIII) as described by the peptide-stimulated kcat ) 1
s-1 (Table 1). The individual steps along this pathway will
need to be defined in the future but will depend on the ability
233
k+1 (µM-1 s-1)
k-1 (s-1)
k+2 (s-1)
k+3 (s-1)
k-3 (µM-1 s-1)
k+4 (µM-1 s-1)
k-4 (s-1)
k+5 (µM-1 s-1)
k+6 (s-1)
k-6 (µM-1 s-1)
FitSim value
exptl value
0.7
7
8
0.5
1
10
5
50
5
5
0.7
7
11a
0.5
0.6
NDb
NDb
NDb
NDb
NDb
a
Values obtained from ref 27. b Values have yet to be experimentally
determined.
to isolate enzyme form VI to study using pre-steady-state
techniques. Peptide could still be interacting with enzyme
form VII to promote ADP release, which would be consistent
with the observations proposed in the steady-state studies.
The details concerning exactly how the peptide is interacting
with the enzyme in this pathway will also need to be further
defined and are thus shown only as “peptide delivery &
cleavage” in Scheme 2. We have previously shown that the
pre-steady-state peptide hydrolysis exhibits lag kinetics (27).
Although no definitive evidence exists, we have proposed
that the lag is an ATP-dependent translocation step (27). In
order to probe the validity of this proposal, the microscopic
rate constants associated with peptide binding, delivery, and
cleavage are currently being investigated.
Previously, we reported a sequential ATP hydrolysis
reaction model to account for the functional nonequivalency
detected in the two ATPase sites in Lon (27). This model
assumed that ATP occupancy at the low-affinity sites
promoted the subsequent hydrolysis of ATP at the highaffinity sites. Further experimentation revealed that the
hydrolysis of the two ATPase sites appeared independent of
one another and that the pre-steady-state burst in ADP
production could be attributed solely to low-affinity ATPase
site activity (28). In light of the results obtained in this work
and in previous single turnover experiments, we have revised
the former kinetic model for the low-affinity ATPase sites
as shown in Scheme 3. To check for consistency between
the revised model and the S2-stimulated ATP hydrolysis
reaction time courses that were reported previously, we
collectively re-fit the data to the revised mechanism by
regression analysis using FitSim (40-42). The results are
summarized in Table 5 and Figure 6. As the burst amplitude
but not the burst rate constants of the time courses varied
with [ATP], we input the burst amplitude values determined
11442 Biochemistry, Vol. 45, No. 38, 2006
Vineyard et al.
are shown. This material is available free of charge via the
Internet at http://pubs.acs.org.
REFERENCES
FIGURE 6: Collective fit of acid-quench ATPase data from ref 27
using FitSim. Simulation of the ATPase mechanism outlined in
Scheme 3 was performed using FitSim. The resulting solid lines
yielded the rate constants summarized in Scheme 3 and Table 5
and were overlaid with the experimental data from ref 27 for ATP
hydrolysis. This demonstrates consistency with the proposed
sequential mechanism. The experimental time courses represent the
hydrolysis of [R-32P]ATP as determined using an acid quench
experiment at (×) 5 µM ATP, (+) 10 µM ATP, (]) 25 µM ATP,
(3) 50 µM ATP, (O) 100 µM ATP, and (0) 200 µM ATP.
previously as the specified enzyme concentration for the
corresponding [ATP] in the fitting process. In Scheme 3,
the enzyme form F represents only the low-affinity ATPase
sites and corresponds to the graphical enzyme for IV depicted
in Schemes 1 and 2. It is discernible from Figure 6 that the
kinetic data overlay well with the fitted time courses and
from Table 5 that the theoretical rate constants agree closely
with those obtained experimentally. It should be noted that
although the hydrolytic activities of the high- and low-affinity
sites are independent of one another, it is possible that the
binding of ATP to the high-affinity sites may still have an
effect on ATP binding to the low-affinity sites such that only
the burst amplitude but not the burst rates of ATP hydrolysis
at the low-affinity sites are affected. The kinetic experiments
reported in this study cannot resolve this issue, and experiments beyond kinetics may be needed to further address this
issue.
ACKNOWLEDGMENT
We thank Hilary Frase for aid in cloning phosphate binding
protein and Jessica Ward for careful reading of the manuscript.
SUPPORTING INFORMATION AVAILABLE
ATP binding and release experiments were performed
using MANT-dATP and MANT-dADP, respectively. The
deoxyribonucleotides were used in order to ensure that the
biphasic nature of the time courses was not a result of
esterification of the fluorophore on the ribonucleotides
MANT-ATP and MANT-ADP. Representative biphasic time
courses of MANT-dATP binding and MANT-dADP release
234
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BI060809+
4602
Biochemistry 2006, 45, 4602-4610
Single-Turnover Kinetic Experiments Confirm the Existence of High- and
Low-Affinity ATPase Sites in Escherichia coli Lon Protease†
Diana Vineyard, Jessica Patterson-Ward, and Irene Lee*
Department of Chemistry, Case Western ReserVe UniVersity, CleVeland, Ohio 44106
ReceiVed NoVember 21, 2005; ReVised Manuscript ReceiVed January 31, 2006
ABSTRACT: Lon is an ATP-dependent serine protease that degrades damaged and certain regulatory proteins
in vivo. Lon exists as a homo-oligomer and represents one of the simplest ATP-dependent proteases
because both the protease and ATPase domains are located within each monomeric subunit. Previous
pre-steady-state kinetic studies revealed functional nonequivalency in the ATPase activity of the enzyme
[Vineyard, D., et al. (2005) Biochemistry 44, 1671-1682]. Both a high- and low-affinity ATPase site has
been previously reported for Lon [Menon, A. S., and Goldberg, A. L. (1987) J. Biol. Chem. 262, 1492114928]. Because of the differing affinities for ATP, we were able to monitor the activities of the sites
separately and determine that they were noninteracting. The high-affinity sites hydrolyze ATP very slowly
(kobs ) 0.019 ( 0.002 s-1), while the low-affinity sites hydrolyze ATP quickly at a rate of 17.2 ( 0.09
s-1, which is comparable to the previously observed burst rate. Although the high-affinity sites hydrolyze
ATP slowly, they support multiple rounds of peptide hydrolysis, indicating that ATP and peptide hydrolysis
are not stoichiometrically linked. However, ATP binding and hydrolysis at both the high- and low-affinity
sites are necessary for optimal peptide cleavage and the stabilization of the conformational change associated
with nucleotide binding.
Lon is an ATP-dependent serine protease functioning to
degrade damaged and certain regulatory proteins in vivo (110). Lon belongs to the ATPases associated with a variety
of cellular activities (AAA+) superfamily, whose members
include ClpAP, ClpXP, ClpCP, and HslUV (11, 12). They
share a conserved Walker A (or P loop) and Walker B motif,
which is associated with nucleotide binding and hydrolysis
(13). Lon represents one of the simplest of the ATPdependent proteases because both the protease and ATPase
domains are located within each monomeric subunit (14, 15).
Crystal structures of portions of the enzyme have been
recently reported and include an inactive mutant of the Lon
protease domain (16-18). This structure shows Lon as a
hexamer organized in a ring with a central cavity, which is
commonly found in other ATP-dependent proteases (11, 13,
17). Although it is known that ATP modulates the protease
activity of Lon (4, 5, 7, 19), mechanistic details concerning
how the binding and hydrolysis of ATP are coordinated with
peptide bond cleavage is not known. However, it has been
shown that ATP binding and hydrolysis do not affect the
oligomeric state of the enzyme (20, 21).
We have previously developed a continuous fluorescent
peptidase assay to monitor the kinetics of peptide cleavage.
Because the inner-filter effect of fluorescence interferes at
high concentrations of 100% fluorescent peptide, we use S3,1
a 10% mixture of fluorescently labeled peptide with its
nonfluorescent analogue (S2) (22). No optical signal from
the peptide is needed when monitoring ATPase activity;
therefore, only the nonfluorescent analogue (S2) is used to
†
This work was support by the NIH Grant GM067172.
* To whom correspondence should be addressed. Telephone: 216368-6001. Fax: 216-368-3006. E-mail: [email protected].
account for the effect of the peptide (23). This 10 amino
acid long (S3 and S2) peptide sequence contains only one
cleavage site and comes from the λN protein, which is a
physiological substrate of Escherichia coli Lon (24). Because
our model peptide (S3 and S2) contains only one Lon
cleavage site and it stimulates ATP hydrolysis, its kinetics
of degradation can be directly attributed to the ATPdependent peptidase reaction rather than polypeptide unfolding or processive peptide cleavage (22, 24).
We have utilized this peptide substrate in steady-state
kinetic and product inhibition studies to establish a minimal
kinetic mechanism for E. coli Lon protease (22). This
mechanism proposed a sequential mechanism for ATP
binding and hydrolysis, which mediated peptide cleavage
presumably through a predicted ATP-dependent translocation
step. The resulting Lon/ATP-bound enzyme form (F) was
distinct from the precatalytic Lon (E). Because the steadystate methods and predicted kinetic model could not address
the microscopic details along the reaction pathway, we
utilized pre-steady-state kinetic techniques to determine the
timing of events as well as individual rate constants.
Previously, we were able to elucidate the timing of ATP
1
Abbreviations: AMPPNP, adenylyl 5-imidodiphosphate; DTT,
dithiothreitol; Abz, anthranilamide; Bz, benzoic acid amide; HBTU,
O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate; HEPES, N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid; KPi,
potassium phosphate; Mg(OAc)2, magnesium acetate; KOAc, potassium
acetate; PEI-cellulose, polyethyleneimine-cellulose; S2, a nonfluorescent analogue of S3 that is degraded by Lon identically as S3 and
is used in the ATPase reactions to conserve the fluorescent peptide
(S3): YRGITCSGRQK(benzoic acid amide) (Bz); S3, a mixed peptide
substrate containing 10% of the fluorescent peptide Y(NO2)RGITCSGRQK(Abz) and 90% S2.
10.1021/bi052377t CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/21/2006
Reproduced with permission from D. Vineyard et al, Biochemistry 45, 4602 (2006).
Copyright 2006 American Chemical Society.
236
High- and Low-Affinity ATPase Sites in Lon
Biochemistry, Vol. 45, No. 14, 2006 4603
Scheme 1: Enzyme Forms Associated with Various Concentrations of
ATPa
a
Form A is a free enzyme containing two different sets of ATPase sites that are represented by gray squares and circles. Form B is formed under
single-turnover conditions when only 500 nM ATP is present. The occupancy of ATP to an enzyme subunit is illustrated by the change in color
from gray to yellow. Form C represents the enzyme form where only the tight sites are occupied by ATP. Form D represents an enzyme form where
both the tight and weak sites are saturated with ATP.
hydrolysis and peptide cleavage in Lon (25), by demonstrating that ATP hydrolysis was occurring before peptide
cleavage during the first turnover of Lon. This study found
that E. coli Lon exhibits lag kinetics in the degradation of
S3 but burst kinetics in ATP hydrolysis. Furthermore, the
ATPase activity of Lon reveals functional nonequivalency
in the subunits of the enzyme, because only 50% of the ATP
bound to Lon is hydrolyzed before peptide cleavage (25).
The observed asymmetry in the ATPase activity could be
attributed to the two different classes of ATP-binding sites
found in E. coli Lon as reported by Menon and Goldberg
(Kd < 1 µM and Kd ∼ 10 µM) (26).
Thus far, kinetic data on the ATPase-dependent degradation of the model peptide S3 by Lon implicates a reaction
model by which the nonequivalent ATPase sites function
cooperatively to modulate the efficiency of peptide cleavage
(25). This model predicts that ATP hydrolysis will occur
with a burst rate constant of ∼12 s-1 at the tight sites of
Lon only when the low-affinity sites are occupied by ATP.
Furthermore, optimal peptide hydrolysis is attained through
the coordinated ATP binding to the low-affinity sites and
hydrolysis at the high-affinity sites. However, this model is
constructed on the basis of kinetic data obtained under pseuofirst-order conditions, where the concentration of ATP is in
excess over Lon. As such, the functional roles of the highand low-affinity ATPase sites could not be independently
examined, and the validity of the proposed model could not
be rigorously tested. To further investigate the cooperative
function of the two kinds of ATPase sites in Lon and their
respective impact on the kinetics of peptide cleavage, we
monitored the ATPase and peptidase activities under limiting
nucleotide concentrations, where only the high-affinity ATPbinding sites of Lon are occupied (Scheme 1). Under these
conditions, although multiple rounds of peptide hydrolysis
occur, the rate constant is 10-fold lower than that obtained
when both ATP-binding sites are occupied under saturating
levels of ATP. Dependent upon the level of ATP saturation,
Lon exhibits two distinct kinetic behaviors in its ATPase
sites, with optimal peptide hydrolysis occurring upon full
occupancy of ATP at both of the sites. Unexpectedly, ATP
hydrolysis at the high-affinity sites is stimulated by peptide
237
or protein substrates and is independent of nucleotide binding
at the low-affinity ATPase sites. Collectively, the data
obtained in this study reveal that peptide cleavage is not
stoichiometrically linked to ATP hydrolysis because multiple
rounds of peptide hydrolysis occur under conditions of
limiting ATP, where only the high-affinity sites are occupied.
We have also shown that the two ATPase sites hydrolyze
ATP at drastically different rates, which are seemingly
unaffected by ATP hydrolysis at the other site. The previously proposed reaction model (25) is therefore revised
accordingly to account for our currently observed data.
MATERIALS AND METHODS
Materials. ATP and casein was purchased from Sigma,
whereas [R-32P]ATP was purchased from Perkin-Elmer or
ICN Biomedical. Fmoc-protected amino acids, Boc-anthranilamide (Abz), Fmoc-protected Lys Wang resin, and Obenzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU) were purchased from Advanced ChemTech
and NovaBiochem. Tris, N-2-hydroxyethylpiperazine-N′ethanesulfonic acid (HEPES), and polyethyleneimine-cellulose (PEI-cellulose) thin-layer chromatography (TLC)
plates were purchased from Fisher.
General Methods. Peptide synthesis and protein purification procedures were performed as described previously (24).
All enzyme concentrations were reported as Lon monomer
concentrations. All reagents are reported as final concentrations.
Double-Filter-Binding Assay. For the high-affinity ATP
site binding experiment, 50 nM [R-32P]ATP was mixed with
0.005-6 µM Lon (27) in 15 µL of 50 mM HEPES at pH
8.0, 5 mM magnesium acetate [Mg(OAc)2], 75 mM potassium acetate (KOAc), and 2 mM dithiothreitol (DTT). A total
of 3 µL of the reactions (performed in triplicate) was spotted
onto a piece of nitrocellulose mounted onto a dot-blot
apparatus (BioRad) with a piece of Immobilon Ny+ below
as described elsewhere (28, 29). All reactions were performed
at least in triplicate. Each spot was washed with 10 µL of
cold reaction buffer 2 times. The radioactive counts at each
spot were quantified by PhosphorImaging using the Packard
4604 Biochemistry, Vol. 45, No. 14, 2006
Vineyard et al.
Cyclone storage phosphor system. The concentration of
bound was determined according to eq 1
[bound] )
(
NCdlu
)
NCdlu + NY+dlu
[[R-32P]ATP]
(1)
where NCdlu is the radioactive count on the nitrocellulose
membrane and NY+dlu is the radioactive count on the
Immobilon Ny+ membrane. The binding parameters were
determined by fitting the data with eq 2 using the nonlinear
regression analysis program Prism (GraphPad) software
version 4
[RL] )
([R] + [L] + Kd) - x([R] + [L] + Kd)2 - 4[R][L]
2[L]
(2)
where [L] is the concentration of [R-32P]ATP, [R] is the
concentration of Lon, [RL] is the concentration of [R-32P]ATP bound to Lon, and Kd is the equilibrium dissociation
constant for ATP bound at the high-affinity site.
Single-TurnoVer ATPase Assays. Single-turnover data for
ATP hydrolysis were measured as described elsewhere (23),
and all reactions were performed at least in triplicate. Briefly,
for the ATPase measurements, each reaction mixture (70 µL)
contained 50 mM HEPES (pH 8.0), 75 mM KOAc, 5 mM
Mg(OAc)2, 5 mM DTT, and 5 or 6 µM Lon monomer. For
the peptide-stimulated ATPase reactions, 500 µM peptide
substrate (S2) was added to each reaction mixture and the
reactions were initiated by the addition of [R-32P]ATP.
Subsequently, 5 µL aliquots were quenched in 10 µL of 0.5
N formic acid at 12 time points (from 0 to 15 min). A 3 µL
aliquot of the reaction was spotted directly onto a PEIcellulose TLC plate (10 × 20 cm), and the plate developed
in 0.75 M potassium phosphate (KPi) buffer (pH 3.4).
Radiolabeled ADP was then quantified using the Packard
Cyclone storage phosphor screen Phosphor imager purchased
from Perkin-Elmer Life Science. To compensate for slight
variations in spotting volume, the concentration of the ADP
product obtained at each time point was corrected using an
internal reference as shown in eq 3
[ADP] )
(
)
ADPdlu
× [ATP]
ATPdlu + ADPdlu
Y ) A exp -kburstt + C
(5)
where t is time in seconds, Y is [ADP] in micromolar, A is
the burst amplitude in micromolar, kburst is the burst rate
constant in s-1, and C is the end point. The observed steadystate rate constants (kss,ATP) were determined by fitting the
data from 600 ms to 3 s with the linear function, Y ) mX +
C, where X is time, Y is [ADP]/[Lon], m is the observed
steady-state rate constant in s-1, and C is the y intercept.
Data fitting was accomplished using the nonlinear regression
program Prism (GraphPad) software version 4.
Tryptic Digestions. Tryptic digest reactions in mixtures
containing 6 µM Lon, 50 mM HEPES (pH 8.0), 5 mM
magnesium acetate, 2 mM DTT, (500 µM S2 peptide, and
either 1 mM ATP, 6 µM ATP, or 500 nM ATP were started
by the addition of 1/50 (w/w) TPCK (N-p-tosyl-L-phenylalanine chloromethyl ketone)-treated trypsin with respect to
Lon. At 0, 2, 4, 20, and 40 min, a 3 µL reaction aliquot was
quenched in 3 µg of soybean trypsin inhibitor (SBTI)
followed by boiling. The quenched reactions were then
resoved by 12.5% SDS-PAGE analysis and visualized with
Coomassie brilliant blue.
RESULTS
(3)
All assays were performed at least in triplicate, and the
kinetic parameters were determined by fitting the time-course
data with a single-exponential eq 4 using the nonlinear
regression program Prism (Graphpad) software version 4
Y ) A exp-kobst + C
Chemical-Quench ATPase ActiVity Assays. The acidquenched time courses for ATP hydrolysis were measured
using a rapid-chemical-quench-flow instrument from KinTek
Corporation as described by Vineyard et al. (25). All
solutions were made in 50 mM HEPES buffer at pH 8.0, 5
mM DTT, 5 mM Mg(OAc)2, and 75 mM KOAc. A 15 µL
buffered solution of 6 µM Lon monomer or 6 µM Lon
preincubated with 6 µM ATP, with and without 500 µM S2
or 20 µM casein, was rapidly mixed with a 15 µL buffered
solution of 100 µM ATP containing 0.01% of [R-32P]ATP
at 37 °C for varying times (0-3 s). The reactions were
quenched with 0.5 N formic acid and then extracted with
200 µL of phenol/chloroform/isoamyl alcohol at pH 6.7
(25:24:1). A 3 µL aliquot of the aqueous solution was spotted
directly onto a PEI-cellulose TLC plate and treated as above.
All assays were performed at least in triplicate, and the
average of those traces was used for data analysis. The burst
amplitudes and burst rates were determined by fitting the
kobs data from 0 to 400 ms with eq 5
(4)
where t is time in seconds, Y is [ADP] in micromolar, A is
the amplitude in micromolar, kobs is the observed rate constant
in s-1, and C is the end point.
Peptidase Methods. Peptidase activity was monitored on
a Fluoromax 3 spectrofluorimeter (Horiba Group) as described previously (22). Assays contained 50 mM HEPES
at pH 8.0, 75 mM KOAc, 5 mM DTT, 5 mM Mg(OAc)2, 1
mM S3 peptide (excitation at 320 nm and emission at 420
nm), 5 or 6 µM Lon, and either ATP or adenylyl 5-imidodiphosphate (AMPPNP) (0-100 µM).
238
Examining Binding of the ATPase Sites in Lon. Although
E. coli Lon contains one ATP-binding domain in each of its
monomeric subunits, the existence of both a high- and lowaffinity ATP-binding site is evident in its oligomeric form
(25, 26). To verify the existence of two different ATPase
sites in Lon under our reaction conditions, we measured the
affinities of Lon for [R-32P]ATP using a filter-binding assay
adapted from the protocols of Jia et al. and Gilbert and
Mackey (27, 28) and Wong and Lohman (29). The half-life
of the complex, where ATP is bound at the low-affinity sites,
can be calculated using the off rate of ATP (Vineyard, D.,
and Lee, I., manuscript in preparation). Because the halflife is on a millisecond time scale, the filter-binding assay
is not an appropriate method for detecting the affinity of
ATP to the low-affinity site. However, the binding to this
site has previously been determined under our reaction
conditions using steady-state kinetic methods (22), and the
resulting affinity agreed with the published value of 10 µM
High- and Low-Affinity ATPase Sites in Lon
Biochemistry, Vol. 45, No. 14, 2006 4605
FIGURE 1: Determining the Kd for the high-affinity ATPase site in
E. coli Lon using an adapted filter-binding assay. To monitor the
binding of [R-32P]ATP to only the high-affinity site, various
concentrations of Lon (0.005-6 µM) were incubated with 50 nM
[R-32P]ATP at 4 °C. The amount of the Lon/[R-32P]ATP complex
formed was quantified by PhosphorImaging of the nitrocellulose
membrane, and the free [R-32P]ATP was quantified by PhosphorImaging of the positively charged Immobilon Ny+ membrane. The
amount of (bound) complex (2) was calculated as described in the
Materials and Methods, and the generated data were fit using a
binding isotherm (eq 2). The resulting Kd value was 0.52 ( 0.096
µM for the high-affinity site.
by Menon and Goldberg (26). The equilibrium dissociation
constant for the high-affinity site was never specifically
determined because of the limit of detection of the previous
assays (Kd < 1µM) (26). Therefore, we utilized the filterbinding assay to better define the binding affinity of Lon to
the high-affinity ATPase site.
To probe the high-affinity site, 50 nM [R-32P]ATP was
incubated with varying amounts of Lon and the resulting
Lon/[R-32P]ATP complex was immobilized onto a nitrocellulose membrane, whereas unbound [R-32P]ATP was
trapped by a positively charged nylon membrane placed
directly below the nitrocellulose filter. The concentration of
Lon was varied rather than [R-32P]ATP to eliminate the high
background generated when the concentration of [R-32P]ATP
is increased because the concentration Lon is held constant
in the nanomolar range. A binding isotherm of the highaffinity ATP site in Lon was generated by quantifying the
amount of 32P immobilized onto the nitrocellulose versus
the nylon membrane as described in the Materials and
Methods. As shown in Figure 1, the binding of ATP at the
high-affinity site was detected with a Kd value of 0.52 (
0.096 µM. Control experiments were performed to ensure
that no [R-32P]ATP hydrolysis was occurring under the
reaction conditions (data not shown). These results are
consistent with the earlier observation reported by Menon
and Goldberg (26) that Lon contains two affinities for ATP,
which differ from one another by approximately 10-fold.
Examining ActiVity of the High-Affinity ATPase Sites of
Lon. Kinetic analyses performed under pseudo-first-order
conditions, where ATP is in excess over the enzyme
concentration, have revealed an apparent functional asymmetry in the ATPase sites (25). We examined the ATPase
activity of the high- and low-affinity sites independently of
one another by manipulating the concentration of nucleotide
such that either just the high-affinity sites or both sets of
sites were occupied. To monitor the ATPase activity at the
high-affinity sites of Lon, single-turnover experiments were
employed. When the concentrations of the reactants are
adjusted such that the Lon concentration (5 µM) is in excess
over limiting ATP (500 nM), only the high-affinity sites are
239
FIGURE 2: Pre-steady-state time courses of ATPase activity of E.
coli Lon under single-turnover conditions. The time courses for
ATP hydrolysis at the high-affinity sites were determined by
incubating 5 µM Lon with 500 nM [R-32P]ATP in the absence (9)
and presence (2) of 500 µM S2 peptide. The reactions were
quenched with acid at the indicated times, and the concentrations
of [R-32P]ADP were determined by TLC followed by PhosphorImaging. The kobs values were determined by fitting the time
courses using a single-exponential equation as described in the
Materials and Methods, yielding observed rate constants of 0.006
( 0.004 and 0.007 ( 0.003 s-1 in the absence and presence of the
S2 peptide, respectively. The inset shows time courses for 500 nM
[R-32P]ATP hydrolysis at the high-affinity sites in the presence of
500 µM S2 peptide at increasing concentrations of Lon: 5 µM
(2), 7 µM ([), and 10 µM (0). The kobs values were determined
by fitting the time courses using a single-exponential equation as
described in the Materials and Methods, yielding observed rate
constants of 0.007 ( 0.001, 0.007 ( 0.001, and 0.006 ( 0.001
s-1, respectively.
occupied by the nucleotide (Kd ) 0.52 µM, see above).
Enzyme forms A-D (Scheme 1) represent different ATPbound states of Lon under the various reaction conditions
used in this study. The proposed enzyme form under limiting
ATP conditions is shown as the enzyme form B in Scheme
1 (17); thus, we can selectively monitor ATP hydrolysis at
only the high-affinity sites. The inset of Figure 2 shows the
hydrolysis of ATP at the high-affinity sites as Lon is
increased (5, 7, and 10 µM). The observed rate constants
range from 0.006 to 0.007 s-1 with a standard deviation of
less than 0.8%. Because the rate constants are identical, the
binding of ATP is not rate-limiting under the single-turnover
reaction conditions employed in the experiment. Furthermore,
as shown in Figure 2, the presence of the S2 peptide does
not influence ATP hydrolysis at the high-affinity sites
because the rate constant for the reaction is 0.006 ( 0.0004
and 0.007 ( 0.0003 s-1 in the absence and presence of a
saturating amount of S2 (500 µM), respectively. This is
consistent with our previous observation that the burst rate
constant associated with ATP hydrolysis under pseudo-firstorder conditions was not affected by the presence of the S2
peptide (25). The rate constant for S2-stimulated ATP
hydrolysis at the high-affinity sites under single-turnover
conditions (kobs ) 0.007 ( 0.0003 s-1), however, is considerably slower than the burst rate obtained when ATP was in
excess over the enzyme concentration (kburst ) 11.3 ( 3.3
s-1) (25). These two experiments differ only by the occupancy of ATP at the low-affinity sites. This implies that,
although one ATP-binding site exists per monomer, two
functionally distinct ATPase sites are evident in the homooligomeric form of Lon.
Because the hydrolysis at the high-affinity ATPase sites
is minimal and ATP hydrolysis is required for optimal
peptide cleavage (23), we questioned whether the catalytic
efficiency of S3 cleavage is coupled with ATP hydrolysis
4606 Biochemistry, Vol. 45, No. 14, 2006
Vineyard et al.
FIGURE 4: Representative E. coli Lon time courses of ATP
hydrolysis at the high-affinity sites. [R-32P]ATP (6 µM) was
incubated with 6 µM monomeric Lon in the absence (9) or presence
(2) of 500 µM S2 peptide and quenched with acid at varying time
points. To see the effect of nucleotide occupation at the low-affinity
sites on the high-affinity site ATP hydrolysis, 100 µM ATP was
added at 1 half-life into the reaction 60 s in both the absence (1)
and presence ([) of 500 µM S2 peptide to saturate the low-affinity
ATPase sites. The time of addition of the 100 µM ATP in traces 1
and [ is indicated by the arrow. As described in the Materials and
Methods, the time courses were fit using the equation Y ) A
exp(-kobs,ATPt) + C, where A is the amplitude, kobs,ATP is the observed
rate constant, and C is the endpoint. The resulting rate constants
are summarized in Table 2. The time points reported are an average
of at least three different trials.
FIGURE 3: S3 hydrolysis by E. coli Lon under limiting nucleotide
conditions. The 5 µM Lon monomer was incubated with 1 mM S3
peptide in the presence of 0, 0.5, and 100 µM ATP and 0.5 and
100 µM AMPPNP. The fluorescence changes associated with
peptide cleavage were converted to product concentrations as
described in the Materials and Methods. The kobs values associated
with each trace are summarized in Table 1.
Table 1: Rate Constants Associated with Peptidase Activity
limiting ATP
stoichiometric ATP
saturating ATP
saturating AMPPNP
[nucleotide] (µM)
kobs,S3 (s-1)
0.5
5
100
100
0.32 ( 0.07
1.52 ( 0.05
2.69 ( 0.30
0.96 ( 0.08
at the high-affinity sites. To address this issue, we monitored
the kinetics of S3 cleavage under single-turnover conditions,
where ATP is limiting (5 µM Lon, 500 nM ATP, and 1 mM
S3). Under these conditions, the predominant enzyme form
is homo-oligomeric Lon, with ATP bound only at the highaffinity sites (enzyme form B in Scheme 1). As shown in
Figure 3, although the rate constant for S3 cleavage at
limiting ATP concentrations (Table 1, kobs,S3 ) 0.32 s-1) is
slower than with saturating ATP, enzyme form D in Scheme
1 (Table 1, kobs,S3 ) 2.69 s-1), Lon is undergoing multiple
rounds of peptide cleavage with only limiting amounts of
ATP. Because no peptide hydrolysis occurs in the presence
of limiting amounts of the nonhydrolyzable ATP analogue,
AMPPNP, (Figure 3) at least one molecule of ATP must be
hydrolyzed for peptide cleavage to occur under these
conditions. Although, nonstoichiometric processing of ATP
and the S3 peptide is observed under saturating (100 µM)
ATP conditions, the single-turnover data much more clearly
demonstrate that ATP and peptide hydrolysis are not
stoichiometric. As previously reported and shown here in
Figure 3 for a comparison to the limiting nucleotide
conditions, saturating amounts of AMPPNP (100 µM)
support S3 hydrolysis at a lower rate than under saturating
ATP conditions (100 µM; Table 1) (22). In this case,
saturating AMPPNP most likely supports slow peptide
hydrolysis because its binding at the low-affinity sites in
addition to not generating ADP from the lack of hydrolysis
is sufficient to lock Lon into an active conformation (22).
Binding and Hydrolysis of ATP at the Tight Sites Are
Independent of Nucleotide Binding and Hydrolysis at the
Weak Sites. To further probe the functional nonequivalency
of the two ATPase sites, the activity of each site was
monitored independently of the other using radiolabeled ATP
as a selective probe. Because the Kd for binding ATP at the
high-affinity sites was 0.52 ( 0.096 µM, the single-turnover
experimental conditions employed above were not sufficient
to saturate those sites. To detect the full effect of ATP
hydrolysis when the high-affinity sites were fully occupied,
240
Table 2: Rate Constants Associated with High-Affinity Site ATPase
Activity
stoichiometric ATP
100 µM ATP chase
100 µM AMPPNP chase
100 µM ADP chase
intrinsic
kobs,ATP (s-1)
S2 stimulated
kobs,ATP (s-1)
0.011 ( 0.001
0.012 ( 0.001
0.010 ( 0.001
0.015 ( 0.001
0.019 ( 0.002
0.017 ( 0.001
0.014 ( 0.001
0.017 ( 0.001
the concentration of [R-32P]ATP was raised to approximately
10-fold excess of the Kd (6 µM), which is stoichiometric to
the amount of Lon in the reaction. Shown as enzyme form
C in Scheme 1, under these conditions, it is assumed that
the high-affinity sites are saturated and the low-affinity sites
are left unoccupied.
The experiment shown in Figure 4 shows the effect on
ATP hydrolysis at the high-affinity sites when the lowaffinity sites were subsequently occupied with unlabeled
nucleotide. To accomplish this, ATP hydrolysis was measured at the high-affinity sites under stoichiometric [R-32P]ATP/Lon conditions (enzyme form C in Scheme 1), while
saturating (100 µM) unlabeled ATP was subsequently added
or chased to occupy the weak-affinity sites 1 min into the
reaction (enzyme form D in Scheme 1). When the experiment
is performed in this manner, the hydrolysis at the highaffinity sites can be monitored for the first half-life of the
reaction and then the effect of nucleotide occupation at the
low-affinity sites on this hydrolysis can subsequently be seen.
To ensure that 60 s was an appropriate time to add saturating
nucleotide, a control experiment was done where the
saturating nucleotide was added at 10 s and no difference
was noted (data not shown). The rate constants associated
with the hydrolysis of [R-32P]ATP at the high-affinity sites
alone as well as after subsequent occupation of the lowaffinity sites with nucleotide are summarized in Table 2.
These experiments were performed in both the presence (S2
stimulated) and absence (intrinsic) of the S2 peptide substrate
to account for effects of any interaction between the ATPase
High- and Low-Affinity ATPase Sites in Lon
Biochemistry, Vol. 45, No. 14, 2006 4607
FIGURE 5: E. coli Lon pre-steady-state chemical-quenched time
courses of ATP hydrolysis at the low-affinity sites. [R-32P]ATP
(100 µM) was incubated with 6 µM monomeric Lon (9) or 6 µM
monomeric Lon preincubated with 6 µM ATP (2) in the presence
of 500 µM S2 peptide as described in the Materials and Methods.
The preincubation of 6 µM Lon with 6 µM ATP presumably
resulted in 6 µM Lon/6 µM ADP, where the high-affinity ATPase
sites were saturated. The reactions were quenched with acid at the
indicated times, and the concentrations of [R-32P]ADP generated
in the reactions were determined by TLC followed by PhosphorImaging. The time courses from 0 to 400 ms were fit with the
equation Y ) A exp(-kburstt) + C, where A is the burst amplitude,
kburst is the observed burst rate constant, and C is the endpoint. The
resulting kburst for Lon (9) was 15.9 ( 0.07 s-1, and the resulting
kburst for 1 Lon/1 ADP (2) was 17.2 ( 0.09 s-1. The kss,ATP values
were obtained by fitting the time courses from 600 ms to 3 s with
a linear function and dividing the slope by the [Lon] in the reaction
and were 0.40 ( 0.021 s-1 for Lon (9) and 0.11 ( 0.014 s-1 for
1 Lon/1 ADP (2). The time points reported here are an average of
at least three different trials.
and peptidase activities. As illustrated in Table 2, the
observed rate constant for ATP hydrolysis at the high-affinity
sites was unaffected by the occupation of the low-affinity
sites 60 s into the reaction with ATP (Figure 4), AMPPNP
(figure not shown), or ADP (figure not shown). Because both
a nonhydrolyzable ATP analogue (AMPPNP) and a product
inhibitor (ADP) were also tested, these data indicate that ATP
hydrolysis at the high-affinity sites is independent of ATP
binding and/or hydrolysis at the low-affinity sites. The rate
constant for ATP hydrolysis at the high-affinity sites was
slightly faster in the presence of the S2 peptide (Table 2)
and casein (data not shown), thus suggesting that communication occurs as a result of the peptide or protein
interacting with the high-affinity ATPase site of Lon.
Peptide and Protein Substrates Stimulate ATP Hydrolysis
at the High-Affinity Sites. In the previous experiment, which
was depicted in Figure 4 for ATP, 100 µM AMPPNP, ATP,
and ADP did not compete out the [R-32P]ATP bound at the
high-affinity sites because the amplitudes of the time courses
were unaffected. The converse experiment could then be
employed to monitor ATP hydrolysis at the low-affinity sites.
In this experiment, Lon (6 µM) was preincubated with a
stoichiometric amount of unlabeled ATP (6 µM; enzyme
form C in Scheme 1). During this preincubation, only the
high-affinity sites are occupied with unlabeled ATP, which
was hydrolyzed to ADP that remains bound at the highaffinity sites. As such, the subsequent hydrolysis of 100 µM
[R-32P]ATP by Lon would directly reflect the ATPase activity
at the low-affinity sites. To ensure that the ATP hydrolysis
at the high-affinity sites, which occurred during the preincubation, was not necessary, the experiment was also
performed where Lon (6 µM) was preincubated with ADP
(6 µM) and the traces were identical (data not shown). Figure
5 shows the hydrolysis of [R-32P]ATP at the low-affinity sites
241
when the high-affinity sites are occupied by unlabeled ADP
(2). Hydrolysis of [R-32P]ATP at the low-affinity sites
exhibited an initial burst in [R-32P]ADP production, which
when fit using eq 6 from the Materials and Methods, yielded
an observed burst rate constant of 17.2 ( 0.09 s-1 (2), which
is comparable to the value of 15.9 ( 0.07 s-1 (9), where
both the high- and low-affinity sites are contributing. As
expected, the rate constant obtained here, 15.9 ( 0.07 s-1
(9), agrees well with the previously determined value of 11.3
( 3.3 s-1 (25). The pre-steady-state time course showing
the hydrolysis of ATP at only the low-affinity ATPase sites
(2) mirrored the pre-steady-state time course reflecting
activity at both sites (9). This would imply that the presteady-state burst in ADP production is coming from only
the low-affinity ATPase sites. As noted in the previous
publication, the ATPase burst activity of Lon is unusual
because it shows half-burst amplitude and the time course
is triphasic (25). The triphasic time course showed a burst
in ADP production followed by an intermediate slow phase,
and then steady-state ATP turnover occurs. The time courses,
therefore, cannot be fit using the classical burst equation
because of the intermediate slow phase. As described in the
Materials and Methods, the time courses are instead split
into the pre-steady-state burst phase, which is fit using a
single-exponential equation, and a linear steady-state phase.
The kinetics of each site has been monitored in the
presence and absence of nucleotide occupation at the other
site, and the occupancy of ADP at the high-affinity sites does
not affect ATP hydrolysis at the low-affinity sites. Therefore,
we have now demonstrated that the ATPase sites hydrolyze
ATP independently of one another and the rate of ATP
hydrolysis at the high-affinity sites is much slower. This
behavior explains the triphasic time courses observed in the
pseudo-first-order experiments as well as the independence
of the burst rate constant on the concentration of ATP, which
have been previously noted (25). Taken together with the
pre-steady-state characterization of peptide cleavage determined previously, we conclude that ATP hydrolysis occurs
at the low-affinity sites prior to S3 cleavage.
Interestingly, there is an observed 4-fold stimulation in
the steady-state rate in the presence of S2 peptide (Figure
5) or the unstructured protein substrate casein (30, 31; data
not shown). This is consistent with the stimulation observed
in the kcat for ATPase activity (23), but it is only observed
when the high-affinity sites are not occupied by ADP (Figure
4, kss,ATP ) 0.40 ( 0.021 s-1, 9; kss,ATP ) 0.11 ( 0.014 s-1,
2). This supports the implication that, although the hydrolysis
at the high-affinity sites is unaffected by nucleotide occupation at the low-affinity sites, it is affected by the presence
of the peptide or protein substrate.
Optimal Peptidase ActiVity Requires ATP Binding and
Hydrolysis at Both Sites. Communication occurring between
the high-affinity ATPase sites and the S2 peptide or protein
substrate has been suggested by the experiments performed
above. Therefore, the catalytic efficiency of S3 cleavage was
examined under stoichiometric Lon/ATP conditions as well
(enzyme form C in Scheme 1). The kinetics of S3 cleavage
were monitored under conditions where the high-affinity sites
were saturated with ATP (5 µM Lon, 5 µM ATP, and 1 mM
S3), and the proposed enzyme form is shown in Scheme 1.
The rate constants for peptide hydrolysis under these
conditions are summarized in Table 1. Because the rate at
4608 Biochemistry, Vol. 45, No. 14, 2006
Vineyard et al.
FIGURE 6: Limited tryptic digestion of Lon in the presence of varying amounts of ATP. Lon in the presence of 500 µM S2 peptide was
digested with a limiting amount of trypsin and quenched at the indicated times with SBTI as described in the Materials and Methods. The
first lane shows the molecular markers in kilodaltons (from top to bottom): 172, 110, 79, 62, 48, 37, 24, and 19.
100 µM ATP (enzyme form D in Scheme 1) is faster than
at 5 µM ATP (enzyme form C in Scheme 1), both ATPase
sites are contributing to the peptidase activity. Optimal
peptide degradation is consequently recovered by the lowaffinity site ATPase activity. This means that the maximal
rate of peptide degradation is attained only when both the
high- and low-affinity sites are saturated with ATP. Because
peptidase activity is slower in the presence of the nonhydrolyzable analogue, AMPPNP, ATP binding as well as
hydrolysis at the low-affinity sites are a necessity for optimal
peptidase activity.
Tryptic Digest Probes the Conformational Change Associated with ATP Binding. Previously (23), we utilized
limited tryptic digestion to probe the functional role of
nucleotide binding to Lon. We detected an adenine-specific
conformational change at saturating amounts of nucleotide.
Although the digestion yielded a pattern of bands ranging
in size from 7 to 67 kDa, the adenine-specific conformational
change was monitored primarily by the detection of a stable
67 kDa fragment. When sequenced, the 67 kDa fragment
included all domains of Lon (ATPase, SSD, and peptidase),
except the amino-terminal region. Of all of the nucleotides
tested in that study, ATP was shown to be the best activator
of the peptidase activity of Lon. Therefore, it was concluded
that the adenine-specific conformational change contributed
to maintaining the optimal catalytic efficiency of S3 cleavage.
In light of the detection of functional nonequivalency in
the ATP binding and hydrolysis activity in Lon, in this study,
we questioned whether the previously observed conformational change upon nucleotide binding could be assigned to
a specific interaction between ATP with either the high- or
low-affinity ATPase sites in Lon. To address this issue, we
subjected 6 µM Lon to limited tryptic digestion in the
presence of limiting (500 nM; enzyme form B in Scheme
1), stoichiometric (6 µM; enzyme form C in Scheme 1), and
excess (1 mM) amounts of ATP (enzyme form D in Scheme
1) to see the effect on the stability of the adenine-specific
conformational change when one or both ATPase sites were
occupied. On the basis of the two Kd values of ATP, we
anticipated that only the tight sites of Lon were occupied by
ATP in the first two cases. Figure 6 shows the Lon fragments
generated over increasing time in the presence of the S2
peptide under conditions of no nucleotide (lanes 2-5),
limiting (500 nM) ATP (lanes 6-9), stoichiometric (6 µM)
242
ATP (lanes 10-12), and saturating (1 mM) ATP (lanes 1315). The digest pattern was identical in the absence of the
S2 peptide (data not shown). In accordance with what was
previously observed, limited tryptic digestion of Lon in the
presence of ATP yields fragments varying from 23 to 67
kDa. As indicated by these data, the 67 kDa fragment is
substantially stabilized only by full occupation of the lowaffinity ATP sites. Because binding and hydrolysis of ATP
at all ATPase sites are also necessary for optimal peptidase
activity in Lon, a correlation between this adenine-specific
conformational change and accessibility for peptide cleavage
could exist.
DISCUSSION
In this study, we utilized kinetic techniques to better
understand the function of the two nonequivalent ATPase
sites in E. coli Lon protease and their coordination with the
protease activity of the enzyme. Despite being a homooligomer, with one ATP-binding site per monomer, Lon
contains two types of ATPase sites that are functionally or
kinetically distinct from one another. As such, the kinetic
characterization of the activities of the ATPase sites is
imperative because they are structurally indistinguishable.
The experiments represented in Figure 4 demonstrate that
ATP hydrolysis at the high-affinity site is unaffected by the
occupation of nucleotide at the low-affinity site, while the
experiments exemplified in Figure 5 show that hydrolysis
of ATP at the low-affinity site is unaffected by the occupation
of nucleotide at the high-affinity site. Therefore, the kinetic
data support the conclusion that the two ATPase sites are
functioning independent of the other. Our kinetic experiments
have also allowed us to examine the coupling of the ATPase
and peptidase activities of Lon protease, which are not yet
fully understood. Mechanistic studies of other enzymes
including Rho protein (32, 33), multidrug-resistance-associated protein (MRP1) (34), P-glycoprotein (PGP) (35), F1ATPase (36), Na+/K+ ATPase (37), topoisomerase II (38,
39), and Rep helicase (40) have also revealed the functional
roles of multiple ATPase sites.
Previously, this lab has constructed a minimal kinetic
model to account for the ATP-dependent S3 cleavage by Lon
using steady-state kinetics (22). Although this model was
constructed assuming monomeric Lon subunits had equivalent ATPase activity, the order of events has been confirmed
High- and Low-Affinity ATPase Sites in Lon
Biochemistry, Vol. 45, No. 14, 2006 4609
by our current pre-steady-state kinetic studies. This minimal
kinetic model predicted ATP hydrolysis to occur prior to
peptide cleavage. Current as well as previous pre-steadystate kinetic studies (25) are consistent with this model
because E. coli Lon exhibits lag kinetics in the degradation
of the model peptide (S3) and burst kinetics in ATP
hydrolysis. The burst kinetics indicate a rapid buildup of a
reaction intermediate with a rate-limiting step following
chemistry in the reaction pathway, while the lag kinetics for
S3 peptide degradation are consistent with a need for an
accumulation of a reaction intermediate prior to peptide
hydrolysis. The burst kinetics for ATP hydrolysis demonstrated triphasic behavior, where only 50% of the Lon
monomer was hydrolyzed in the duration of the lag in
peptidase activity. Although this unusual behavior suggested
the contribution of two ATPase activities, it could not
separate the two.
The present study has confirmed the existence of two
ATPase sites in Lon and that their activities are independent
of one another. We have now demonstrated with additional
pre-steady-state experiments that the ATP bound to the lowaffinity sites is hydrolyzed during the lag in peptide cleavage
with a burst rate constant of 17.2 ( 0.09 s-1. The peptide is
concomitantly cleaved with a rate constant of 2.69 ( 0.30
s-1, while the ATP bound at the high-affinity sites is slowly
being hydrolyzed with an observed rate constant of 0.019 (
0.002 s-1. The previously determined minimal kinetic model
also predicted a Lon/ATP-bound “F” form of the enzyme
following peptide cleavage, which could undergo multiple
rounds of peptide hydrolysis before reverting back to the
free enzyme (22). This is consistent with our data in Figure
3, which demostrate that peptide and ATP hydrolysis are
not stoichiometrically linked. It is still not clear how the
enzyme turns over once it reaches the “F” form. Our revised
mechanism for the ATPase activity in Lon predicts that the
two ATPase sites are hydrolyzing ATP sequentially but are
not communicating. However, binding and hydrolysis of ATP
at both the high- and low-affinity ATPase sites are necessary
for optimal peptide or protein degradation. The energy
generated from ATP hydrolysis at the low-affinity sites could
be used to translocate the protein substrate or may be used
to induce a conformational change in the oligomer that
facilitates protein cleavage. Therefore, the development of
an effective continuous assay to measure protein degradation
is currently underway to determine how the high- and lowaffinity ATPase sites are contributing to protease activity.
Because the protease activity is ultimately limited by
turnover of ATP hydrolysis, further kinetic characterization
needs to be performed to delineate the ATPase mechanism.
We and others (4) have previously suggested that ADP
release was the rate-limiting step along the reaction pathway
because it is a potent inhibitor of peptidase activity (Ki,ADP
) 0.3 µM) (22). The burst kinetics associated with ATP
hydrolysis would be consistent with this, because they
indicated that a step following chemistry is rate-limiting.
However, the rapid-quench experiment shown in Figure 5
showed that ATP did not compete out ADP bound to the
high-affinity sites of Lon, suggesting that ADP is perhaps
only inhibiting at the high-affinity sites. Experiments to
determine other microscopic rate constants along the ATPase
reaction pathway including ADP release are currently
underway to determine the rate-limiting step of the reaction.
243
ACKNOWLEDGMENT
We thank Dr. Anthony Berdis for his assistance and careful
reading of this manuscript.
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BI052377T
Biochemistry 2005, 44, 1671-1682
1671
Monitoring the Timing of ATP Hydrolysis with Activation of Peptide Cleavage in
Escherichia coli Lon by Transient Kinetics†
Diana Vineyard,‡ Jessica Patterson-Ward,‡ Anthony J. Berdis,§ and Irene Lee*,‡
Department of Chemistry and Department of Pharmacology, Case Western ReserVe UniVersity, CleVeland, Ohio 44106
ReceiVed June 30, 2004; ReVised Manuscript ReceiVed October 14, 2004
ABSTRACT: Escherichia coli Lon, also known as protease La, is an oligomeric ATP-dependent protease,
which functions to degrade damaged and certain short-lived regulatory proteins in the cell. To investigate
the kinetic mechanism of E. coli Lon protease, we performed the first pre-steady-state kinetic
characterization of the ATPase and peptidase activities of this enzyme. Using rapid quench-flow and
fluorescence stopped-flow spectroscopy techniques, we demonstrated that ATP hydrolysis occurs before
peptide cleavage, with the former reaction displaying a burst and the latter displaying a lag in product
production. The detection of burst kinetics in ATP hydrolysis is indicative of a step after nucleotide
hydrolysis being rate-limiting in ATPase turnover. At saturating substrate concentrations, the lag rate
constant for peptide cleavage is comparable to the kcat of ATPase, indicating that two hydrolytic processes
are coordinated during the first enzyme turnover. The involvement of subunit interaction during enzyme
catalysis was detected as positive cooperativity in the binding and hydrolysis of substrates, as well as
apparent asymmetry in the ATPase activity in Lon. When our data are taken together, they are consistent
with a reaction model in which ATP hydrolysis is used to generate an active enzyme form that hydrolyzes
peptide.
Escherichia coli Lon, also known as protease La, is an
oligomeric ATP-dependent protease, which functions to
degrade damaged and certain short-lived regulatory proteins
in the cell (1-10). In Vitro this enzyme is capable of
degrading polypeptides in the presence of certain nonhydrolyzable ATP analogues. However, optimal protein degradation does require ATP hydrolysis (3, 6, 11). Lon
represents one of the simplest forms of ATP-dependent
proteases because both the ATPase and the protease domains
are located within a single monomeric subunit (12, 13).
Although a complete crystal structure of active Lon has
yet to be determined, some structural clues to the mechanism
of Lon are available. The crystal structure of a proteolytically
inactive Lon mutant lacking an ATPase domain reveals that
Lon is hexameric and contains a central cavity commonly
found in the ATPase and protease subunits of other ATPdependent proteases (14). Additionally, the structure of the
R subdomain of the ATPase subunit of Lon has been
determined (15). This monomeric subdomain constitutes the
last 25% of the carboxyl-terminal region of the ATPase
domain and lacks the conserved Walker motifs found in
ATP-binding proteins. On the basis of these reports, Lon
appears to be structurally similar to other ATP-dependent
proteases such as the bacterial homologue of the proteasome,
HslUV (16, 17). Whether and how ATP binding and
hydrolysis are coupled to peptide cleavage are key inquiries
in understanding the mechanism of the enzyme.
†
This work was support by the NIH Grant GM067172.
* To whom correspondence should be addressed. Telephone: 216368-6001. Fax: 216-368-3006. E-mail: [email protected].
‡
Department of Chemistry.
§
Department of Pharmacology.
The coupling of ATP binding and hydrolysis to peptide
cleavage is a key question in mechanistic studies of Lon
protease. On the basis that ADP inhibits the ATPase and
the protease activities and nonhydrolyzable ATP analogues
support peptide cleavage, an ADP/ATP exchange mechanism
has been proposed (3, 6, 18-21). However, it is not known
whether peptide cleavage occurs before or after ATP
hydrolysis. To evaluate the functional relationship between
ATP hydrolysis and peptide cleavage, we developed a
fluorescent peptide substrate whose degradation by Lon
exhibits ATPase dependency as observed in the endogenous
protein substrates of Lon (11, 22). This defined model peptide
substrate containing 10% of the fluorescent peptide Y(NO2)RGITCSGRQK(Abz) and 90% S2 (S3)1 and its nonfluorescent analogue (S2) were previously used as simplified mimics
of the E. coli Lon substrate, the λ N protein, to demonstrate
that kcat but not Km of the peptide cleavage is dependent on
ATP hydrolysis (11). In addition, the kcat of peptide cleavage
is higher when hydrolyzable nucleotides rather than nonhydrolyzable ATP analogues are used as activators. During
peptide cleavage, the nucleotide hydrolysis activity of Lon
1
Abbreviations: AMPPNP, adenylyl 5-imidodiphosphate; DTT,
dithiothreitol; Abz, anthranilamide; Bz, benzoic acid amide; NO2, nitro;
HBTU, O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol; HEPES, N-2hydroxyethylpiperazine-N′-ethanesulphonic acid; Mg(OAc)2, magnesium acetate; KOAc, potassium acetate; PEI-cellulose, polyethyleneimine-cellulose; λ N, also known as the λ N protein, a λ phage protein
that allows E. coli RNA polymerase to transcribe through termination
signals in the early operons of the phage; S2, a nonfluorescent analogue
of S3 that is cleaved by Lon in the same manner as S3:YRGITCSGRQK(benzoic acid amide) (Bz); S3, a mixed peptide substrate
containing 10% of the fluorescent peptide Y(NO2)RGITCSGRQK(Abz)
and 90% S2.
10.1021/bi048618z CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/11/2005
Reproduced with permission from D. Vineyard et al, Biochemistry 44, 1671 (2005).
Copyright 2005 American Chemical Society.
245
1672 Biochemistry, Vol. 44, No. 5, 2005
Vineyard et al.
is also elevated, suggesting a coupling between the two
hydrolytic activities (23). Because both peptides were
degraded by E. coli Lon in an identical manner, the S3 and
S2 peptide could be used as substrates to monitor the
peptidase and ATPase activity of Lon, respectively (11).
While initial velocity and product inhibition studies
identified which enzyme forms predominate under certain
reaction conditions, they could not evaluate whether ATP
hydrolysis precedes or follows peptide cleavage (11). This
question, however, can be answered by measuring the rates
of ATP and peptide hydrolysis in the pre-steady state. If ATP
hydrolysis occurs before peptide cleavage, its rate constant
should be higher than that for peptide cleavage. In this study,
we utilized rapid chemical-quench-flow and fluorescence
stopped-flow techniques to monitor pre-steady-state time
courses of the two reactions to correlate the timing of ATP
hydrolysis with peptide cleavage, thereby establishing the
sequence of events occurring along the reaction pathway.
In addition, despite its existence as a homooligomer, Lon
exhibits two different affinities for ATP (Kd ) 10 µM and
Kd < 1 µM) (19); the mechanism by which these two kinds
of ATPase sites affect proteolysis are not known. Therefore,
by determining the pre-steady-state kinetics of peptide and
ATP hydrolysis at low (<10 µM) versus high (100 µM) ATP
concentrations, we can evaluate how the binding and/or
hydrolysis of the nucleotide at the respective ATPase site
coordinates with the first turnover of peptide cleavage.
MATERIALS AND METHODS
Materials. ATP was purchased from Sigma, whereas
[R-32P]ATP was purchased from Perkin-Elmer or ICN Biomedical. Fmoc-protected amino acids, Boc-anthranilamide
(Abz), Fmoc-protected Lys Wang resin, and O-benzotriazoleN,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU)
were purchased from Advanced ChemTech and NovaBiochem. 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris),
N-2-hydroxyethylpiperazine-N′-ethanesulphonic acid (HEPES),
and polyethyleneimine-cellulose (PEI-cellulose) TLC plates
were purchased from Fisher.
General Methods. Peptide synthesis and protein purification procedures were performed as described previously (22).
All enzyme concentrations are reported as Lon monomer
concentrations. All reagents are reported as final concentrations.
Acid-Quench Peptidase Assay. Assays were performed at
37 °C and contained 50 mM Tris-HCl (pH 8.1), 5 mM
magnesium acetate (Mg(OAc)2), 5 mM dithiothreitol (DTT),
800 µM S3 peptide substrate (containing 50% of the
fluorescent peptide, S1, and 50% S2, the nonfluorescent
analogue of S1 that is degraded by Lon in an identical
manner as S1), and 1 µM E. coli Lon monomer. The reaction
was initiated by the addition of 500 µM ATP, and 10 µL
aliquots were quenched with 58 µL of 0.5 N HCl at 0, 0.5,
1, 2, 3, and 5 min. After trichloroacetic acid precipitation,
which removed Lon, the reaction mixtures were neutralized
with 1 M Tris/2 N NaOH to pH 8 and the fluorescence
(excitation of 320 nm and emission of 420 nm) of the
solution was measured using a Fluoromax 3 spectrofluorimeter (Horiba Group).
Pre-steady-State Time Courses of S3 CleaVage by Fluorescent Stopped Flow. Pre-steady-state experiments were
246
performed on a KinTek Stopped Flow controlled by the data
collection software Stop Flow version 7.50 β. The sample
syringes were maintained at 37 °C by a circulating water
bath. Syringe A contained 5 µM Lon monomer, with variable
concentrations of the peptide substrate S3 (from 5 to 500
µM), 5 mM Mg(OAc)2, 50 mM Tris-HCl (pH 8.1), and 5
mM DTT. Syringe B contained varying ATP (1-500 µM).
Peptide cleavage was detected by an increase in fluorescence
(excitation of 320 nm and emission of 420 nm) following
rapid mixing of syringe contents in the sample cell. The
baseline fluorescence was normalized to zero, and the data
shown are a result of averaging at least four traces. The
concentration of hydrolyzed peptide was calibrated by
determining the maximum fluorescence generated per micromolar peptide because of complete digestion by trypsin
under identical reaction conditions in the stopped-flow
apparatus. The averaged time courses were fit with eq 1
Y ) A exp-klagt + Vsst + C
(1)
where t is time in seconds, Y is the concentration of
hydrolyzed peptide S3 in micromolar, A is the amplitude of
the reaction, klag is the pre-steady-state rate constant in per
seconds, Vss is the steady-state rate in units of micromolar
product per second, and C is the endpoint. The Vss value can
be converted to a first-order rate constant (kss in the unit of
per seconds) by division with the enzyme concentration (24).
Equation 1 is the general function that quantifies a biphasic
time course. When Y ) 0 at t ) 0, C ) -A and eq 1 becomes
Y ) -A + A exp-klagt + Vsst
such that
Y ) -A(1 - exp-klagt) + Vsst
When A is defined as Vss - Vi/klag, eq 1 becomes equivalent
to the equation defining hysteresis (25, 26), where Vi is the
rate corresponding to the initial phase of the time course.
Data Processing. Plots displaying sigmoidal behavior were
fit with eq 2 where k is the observed rate constant of the
k)
kmax[S]n
K′ + [S]n
(2)
reaction being monitored, kmax is the maximum rate constant
(referred to as kcat,S3 for kss,S3 data, klag,S3 for klag data, and
kcat,ATP for kss,ATP data), [S] is the variable substrate, K′ is the
Michaelis constant for S, and n is the Hill coefficient. The
Ks is calculated from the relationship log K′ ) n log Ks,
where Ks is the [S] required to obtain 50% of the maximal
rate constant of the reaction. Plots displaying hyperbolic
behavior, i.e., when the Hill coefficient in eq 2 becomes 1,
were fit using eq 3 where k is the observed rate constant,
k)
kmax[S]
Ks + [S]
(3)
kmax is the maximum rate constant (referred to as kcat,S3 for
kss,S3 data, klag,S3 for klag data, and kcat,ATP for kss,ATP data), [S]
is the variable substrate, and Ks is the [S] required to obtain
50% of the maximal rate constant of the reaction. The Ks
Kinetic Mechanism of ATP Hydrolysis and Peptide Cleavage
value was referred to as Km,S3 when S3 hydrolysis was
monitored at varying S3 concentrations. Alternatively, the
Ks value was referred to as Km,ATP when ATP hydrolysis was
monitored at varying ATP concentrations.
Chemical-Quench ATPase ActiVity Assays. The pre-steadystate time courses for ATP hydrolysis were measured using
a rapid chemical-quench-flow instrument from KinTek
Corporation. All solutions were made in 50 mM HEPES
buffer at pH 8.1, 5 mM DTT, 5 mM Mg(OAc)2, and 75 mM
potassium acetate (KOAc). A 15 µL buffered solution of 5
µM Lon monomer, with and without 500 µM S2 or 10 µM
casein, was rapidly mixed with a 15 µL buffered solution of
ATP containing 0.01% of [R-32P]ATP at 37 °C for varying
times (0-3 s) before quenching with 0.5 N formic acid and
then extracted with 200 µL of phenol/chloroform/isoamyl
alcohol at pH 6.7 (25:24:1). A 3 µL aliquot of the aqueous
solution was spotted directly onto a PEI-cellulose TLC plate
(10 × 20 cm), and the plate was developed in 0.75 M
potassium phosphate buffer (pH 3.4) to separate ADP from
ATP. The relative amount of radiolabeled ADP and ATP at
each time point was quantified by a Cyclone Phosphor
imager (Perkin-Elmer Life Science). To compensate for the
slight variations in spotting volume, the concentration of the
ADP product obtained at each time point was corrected for
using an internal reference as shown in eq 4. All assays were
[ADP] )
(
)
ADPdlu
[ATP]
ATPdlu + ADPdlu
(4)
performed at least 3 times, and the average of those traces
was used for data analysis. The burst amplitudes and burst
rates were determined by fitting the kobs data from 0 to 400
ms with eq 5. where t is time in seconds, Y is [ADP] in
Y ) A exp-kburstt + C
(5)
micromolar, A is the burst amplitude in micromolar, kburst is
the burst rate constant in per seconds, and C is the end point.
The observed steady-state rate constants (kss,ATP) were
determined by fitting the data from 600 ms to 1.8 s with the
linear function, Y ) mX + C, where X is time, Y is [ADP]/
[E], m is the observed steady-state rate constant in per
seconds, and C is the y intercept. Data fitting was accomplished using the nonlinear regression program KaleidaGraph (Synergy).
Pulse-Chase ATPase ActiVity Assays. The pre-steady-state
time courses of ATP hydrolysis were also measured using a
pulse-chase experiment on the rapid quench. Lon ((0.5 mM
S2) was rapidly mixed with radiolabeled ATP at 37 °C for
0-1.8 s, followed by a 10 mM unlabeled ATP chase for 60
s before quenching with 0.5 N formic acid. The amount of
ADP produced at each time point was quantified as described
in the chemical-quench assay (see above). The burst amplitude (A) and burst rate constant (kburst) were determined from
the time courses by fitting the data from 0 to 400 ms with
eq 5.
Filter Binding Assay. A total of 2-5 µL of a 35 µM stock
of Lon was incubated with 10 µM [R-32P]ATP in 30 µL of
50 mM HEPES at pH 8.1, 5 mM Mg(OAc)2, 75 mM KOAc,
and 2 mM DTT at 37 °C for 20 min to convert all ATP to
ADP. The reactions were then chilled on ice, and 3 µL of
the reactions (performed 3 times) was spotted onto a piece
of nitrocellulose mounted onto a dot-blot apparatus (BioRad)
247
Biochemistry, Vol. 44, No. 5, 2005 1673
FIGURE 1: Detection of ATP-dependent S3 cleavage by a discontinuous acid-quench assay. Lon (1 µM monomer) was incubated
with 800 µM S3, and reaction aliquots were quenched at the
indicated times. The fluorescence signals associated with peptide
cleavage were measured and plotted against their corresponding
reaction time points. The experiment was performed 2 times, and
the averaged data were plotted. The data were fit with eq 1 to yield
a lag rate constant of 1.4 s-1.
as described by Gilbert and Mackey (24). Each spot was
washed with 10 µL of cold buffer and dried under vacuum
for 30 min. In the absence of vacuum, the nitrocellulose was
spotted with 2 µL of each reaction and then air-dried. The
radioactive counts at each spot were quantified by PhosphorImaging.
RESULTS
Pre-Steady-State Kinetic Analysis of S3 CleaVage. Previously, we demonstrated that the pre-steady-state time course
of S3 cleavage could be monitored by stopped-flow fluorescence spectroscopy (11). Using ATP as the activator, we
detected lag kinetics in S3 cleavage. The lag phase is
lengthened by 7-fold when adenylyl 5-imidodiphosphate
(AMPPNP), a non-hydrolyzable ATP analogue, was used
as an activator. Because the cleavage of S3 peptide generates
a fluorescent signal as a result of the separation of the
fluorescent quencher, 3-nitrotyrosine, from the fluorescent
donor, anthranylamide, the lag in S3 cleavage could be
attributed to a slow step prior to peptide bond cleavage or
to the slow dissociation of the donor from the quencher
because both hydrolyzed peptides remain bound to the active
site of the enzyme. To determine if slow dissociation of
peptide products caused the lag, we monitored the time
course of 800 µM S3 cleavage by 1 µM monomeric Lon
using a discontinuous acid-quench assay. An aliquot of the
reaction was quenched with HCl at the times indicated in
Figure 1, and the fluorescence intensity at each time point
was measured to yield the time course for S3 cleavage.
Despite acid denaturation that released hydrolyzed peptides
from Lon, the lag phase remained in the fluorescence time
course of S3 cleavage. This result indicates that the separation
of the hydrolyzed peptide from Lon does not contribute to
the observed lag phase of the reaction. Furthermore, a lag
rate constant of 1.4 ( 0.6 s-1 was obtained by fitting the
data with eq 1, which is a general function used to determine
the rate constant (k) associated with a single-exponential
phase followed by a steady-state phase, and is related to the
hysteresis equation as described in the Materials and
Methods. This observed value is 2-fold higher than that
obtained previously using stopped-flow spectroscopy (11)
and may be attributed to a difference in the detection methods
1674 Biochemistry, Vol. 44, No. 5, 2005
Vineyard et al.
FIGURE 2: Stopped-flow analysis of ATP-dependent S3 cleavage
by E. coli Lon. (A) ATP (500 µM) was incubated with 5 µM
monomeric Lon in the presence of 5 (1), 10 (O), 25 (]), 50 (×),
100 (+), 200 (4), and 500 (b) µM S3. The inset zooms in A to
show the lag for one peptide hydrolyzed per active site. The
fluorescence changes associated with peptide cleavage were
converted to product concentrations as described in the Materials
and Methods. Each time course shown is an average of 4 traces.
The values on the right y axis represent the concentration of peptide
hydrolyzed, whereas the values on the left y axis represent the mole
equivalent of peptide digested by each Lon monomer. (B) S3 (500
µM) was digested by 5 µM Lon monomer in the presence of 1
(b), 5 (0), 10 (]), 50 (×), 100 (+), 200 (4), and 500 (O) µM
ATP. The inset zooms in B to show the lag for one peptide
hydrolyzed per active site. Each time course shown is an average
of at least 4 traces. The right y axis shows the amount of peptide
hydrolyzed, whereas the left x axis shows the mole equivalent of
peptide cleavage by each Lon monomer.
(a continuous versus discontinuous assay). Despite the slight
variation in the lag rate constants, both the stopped-flow and
the acid-quenched reactions displayed lag kinetics, indicating
that the dissociation of the peptide products from Lon did
not contribute to the lag phase.
To determine the stoichiometry of S3 cleavage, we
generated a calibration curve by measuring the fluorescence
changes associated with the complete degradation of known
concentrations of S3 by trypsin in a stopped-flow apparatus.
This technique allows us to accurately define the amount of
peptide cleaved during the time courses for S3 cleavage.
Experiments were performed at 500 µM ATP with several
fixed levels of S3 (5-500 µM, Figure 2A) as well as a fixed
248
FIGURE 3: Steady-state kinetics of ATP-dependent S3 cleavage by
5 µM E. coli Lon monomer. The kss,S3 values were obtained by
dividing the steady-state rates of the reactions by [Lon] as described
in the Materials and Methods. (A) Steady-state rates of S3 cleavage
(Vss) were obtained by fitting the stopped-flow time courses of
peptide cleavage at varying [S3] with eq 1 (Materials and Methods).
The data presented in this plot were best-fit with eq 2, and the
kinetic parameters obtained were kcat,S3 ) 5.5 ( 0.8 s-1, Km,S3 )
188 ( 148 µM, and n ) 1.23 ( 0.2 as summarized in Table 1. (B)
Steady-state rates of S3 cleavage (Vss) were obtained by fitting the
stopped flow-time courses of peptide cleavage at varying [ATP]
with eq 1 (Materials and Methods). The data presented in this plot
were best-fit with eq 3, and the kinetic data obtained from the fit
were kcat,S3 ) 4.2 ( 0.1 s-1 and KATP ) 9.7 ( 0.9 µM as
summarized in Table 1.
concentration of S3 (500 µM) and several fixed levels of
ATP (1-500 µM, Figure 2B). As shown in parts A and B
of Figure 2, a lag in peptide hydrolysis was observed in all
of the time courses. The lag rate constants (klag) and the
observed steady-state rate constants of peptide cleavage
(kss,S3) were obtained by fitting each time course with eq 1.
The plots of kss,S3 as a function of S3 and ATP concentrations
are shown in parts A and B of Figure 3, and the data were
fit with eqs 2 and 3, respectively, to yield the kcat,S3 (the
maximum rate constant for peptide cleavage), KmS3, KATP,
and Hill coefficients (n) as summarized in Table 1. The
steady-state kinetic parameters determined from this study
(using 5 µM enzyme monomer) agree well with those
determined previously at 125 nM Lon monomer (kcat,S3 )
7.7 s-1; KmS3 ) 85 µM, and KATP ) 7.2 µM) (11), indicating
that these kinetic parameters are independent of the enzyme
concentration under the conditions examined. Because presteady-state lag kinetics could be attributed to the binding
of substrates at low concentrations being rate-limiting, we
measured the klag of S3 cleavage at increasing peptide or
ATP concentrations. As shown in parts A and B of Figure
4, the dependence of klag toward S3 and ATP concentrations
reaches saturation with a maximum klag,S3 value. The data
were best-fit with eqs 2 and 3, respectively, to yield the
kinetic parameters klag,S3, KS3, and KATP as summarized in
Kinetic Mechanism of ATP Hydrolysis and Peptide Cleavage
Biochemistry, Vol. 44, No. 5, 2005 1675
-1
Table 1: Parameters Obtained from Pre-steady-State Kinetic
Characterization of ATP-Dependent S3 Cleavage by E. coli Lon
kcat,S3
Km,S3
n
klag,S3
KS3
KATP
0.5 mM ATP
vary [S3]
0.5 mM S3
vary [ATP]
5.5 ( 0.8 s-1 a
188 ( 148 µM a
1.23 ( 0.2a (1.7 ( 0.2)c
0.88 ( 0.07 s-1 c
67 ( 34 µMc
NA
4.2 ( 0.1 s-1 b
NA
NA
1.14 ( 0.06 s-1 d
NA
9.7 ( 0.9b µM (7.2 ( 1.9 µM)c
a
These values were obtained by fitting the steady-state kinetic data
shown in Figure 3A with eq 2. b These values were obtained by fitting
the steady-state kinetic data shown in Figure 3B with eq 3. c These
values were obtained by fitting the lag rate constants obtained from
the stopped-flow time courses of S3 cleavage shown in Figure 4A with
eq 2. d NA ) not available.
FIGURE 4: Substrate dependency of the lag rate constants of S3
cleavage. (A) Lag rate constant for S3 peptide degradation by 5
µM monomeric Lon at varying S3 was determined by fitting the
stopped-flow time courses of peptide cleavage as shown in Figure
2A with eq 1. The data were collectively fit with eq 3 to yield a
maximal klag,S3 ) 0.88 s-1, KS3 ) 67 µM, and n ) 1.7 ( 0.2. The
error bars represent the experimental deviations among the different
trials. Each data point was obtained from the average of three
independent experiments, with each experiment containing at least
four stopped-flow traces. (B) Lag rate constant for S3 peptide
degradation by 5 µM monomeric Lon at varying ATP was
determined by fitting the stopped-flow time courses of peptide
cleavage as shown in Figure 2B with eq 1. The data were
collectively fit with eq 2 to yield a maximal klag,S3 ) 1.14 s-1 and
a KATP ) 7.2 ( 1.9 µM. The error bars represent the experimental
deviations among the different trials. Each data point was obtained
from the average of three independent experiments, with each
experiment containing at least four stopped-flow traces.
Table 1. The maximum lag rate constant for S3 cleavage
(klag,S3), determined at varying S3 and varying ATP concentrations, was 0.88 ( 0.07 and 1.14 ( 0.06 s-1, respectively.
These values are in close agreement with the lag rate constant
249
of 0.76 s determined previously by fitting the fluorescent
time courses with the hysteresis equation (11), which is a
specific form of eq 1 (see the Materials and Methods) used
to characterize enzymes responding slowly to a change in
the ligand concentration (25, 26). At high S3 or ATP
concentrations, substrate binding no longer limits the rate
of the first peptidase turnover, and thus, the maximum klag,S3
value directly reflects the rate constant for the build-up of
at least one reaction intermediate that leads to peptide
hydrolysis at the active site of the enzyme. Furthermore, the
fit of the klag,S3 dependence on S3 or ATP concentrations to
eq 2 (for S3) or eq 3 (for ATP) provided the apparent Kd
values of the respective substrate (KS3 and KATP in Table 1).
The KS3 value (67 ( 34 µM) is comparable to the Km of S3
cleavage (188 ( 148 µM), whereas KATP (9.7 ( 0.9 µM) is
in close agreement with the weak affinity ATPase site in E.
coli Lon, which is 10 µM (17).
Chemical-Quench Analysis of ATP Hydrolysis. Because
the kcat of S3 cleavage is enhanced by nucleotide hydrolysis
(11, 21) and the kcat of ATP hydrolysis is stimulated by the
peptide or protein substrate (3, 21, 23), it is very likely that
the two hydrolytic processes are coupled through a common
enzyme intermediate. To begin identifying such an intermediate, we measured the time courses of the first turnover of
ATP hydrolysis in the absence and presence of the peptide
or protein substrate using the rapid chemical-quench technique. Lon (5 µM monomer) was preincubated with 500 µM
S2, the nonfluorescent analogue of S3 that was degraded by
Lon identically as S3 (5× KmS3, Table 1, see ref 11), and
rapidly mixed with 100 µM ATP (10× KATP, Table 1) prior
to quenching with formic acid at times between 0 and 3 s.
The time courses for ATP hydrolysis measured in the absence
and presence of casein (protein substrate) were also determined (Figure 5A) for comparison. All three time courses
showed an identical burst in ADP production within the first
200 ms of the reaction, followed by at least one slower phase
in product formation. The detection of a pre-steady state burst
in the acid-quench experiments is indicative of the ratelimiting step occurring after ATP hydrolysis. Although the
S2 peptide is smaller than casein and contains only one Lon
cleavage site, it stimulates ATP hydrolysis like the protein
substrate, thereby indicating that both substrates share
identical mechanisms in ATPase stimulation. The data in
Figure 5A were initially fit with eq 1 (with klag becoming
kburst), which characterizes a single-exponential burst phase
followed by a linear steady-state turnover rate of the reaction.
As shown in Figure 5B and the inset, the data obtained during
the first 400 ms of the time courses showed a poor fit with
eq 1 and the burst amplitudes were also significantly
underestimated. To better evaluate the burst amplitudes as
well as the burst rates of the reactions, we fit the data within
the first 400 ms of the time courses, which consisted of a
burst and a relatively constant transition phase in ADP
formation, with eq 5 to yield the values summarized in Table
2. As shown in Table 2 and parts A and C of Figure 5, the
burst amplitudes as well as the burst rate constants for the
three time courses are comparable but the steady-state rates
of the intrinsic versus stimulated ATPase reactions differ.
The steady-state phase of the time courses (from 600 ms to
1.8 s) were fit with a linear function to yield the steadystate turnover numbers of 0.23 ( 0.02, 0.69 ( 0.01, and
0.67 ( 0.03 s-1, corresponding to the intrinsic, S2-stimulated,
1676 Biochemistry, Vol. 44, No. 5, 2005
Vineyard et al.
FIGURE 5: Pre-steady time courses of ATP hydrolysis by E. coli
Lon. (A) [R-32P]ATP (100 µM) was incubated with 5 µM
monomeric Lon in the absence (+) and presence of 500 µM S2
(0) or 10 µM casein (]), and the reactions were quenched with
acid at the indicated times. The concentrations of [R-32P]ADP
generated in the reactions were determined by TLC followed by
PhosphorImaging as described in the Materials and Methods. The
values on the y axis were obtained by dividing [ADP] produced
by 5 µM Lon, which reflects the mole equivalent of ADP produced
per Lon monomer. (B) [R-32P]ATP (200 µM) was incubated with
5 µM monomeric Lon in the presence of 500 µM S2. The time
points were obtained by quenching the reactions with acid at the
indicated times, and the resulting time course for ADP production
was fit with the equation Y ) A exp(-kobst) + Vsst + C, where A
is the burst amplitude, kobs is the observed burst rate constant, Vss
is the steady-state rate, and C is the endpoint. The inset shows the
fit of the data spanning 0-400 ms. (C) Time points from 0 to 400
ms were fit with eq 5 (Materials and Methods) to yield burst
amplitudes for intrinsic, casein-stimulated, and S2-stimulated time
courses of 1.92 ( 0.12, 1.8 ( 0.2, and 2.06 ( 0.15 µM,
respectively, as summarized in Table 2. The burst rate constants
were 7.6 ( 1.3, 12.2 ( 3.2, and 6.5 ( 1.4 s-1, respectively, which
are also summarized in Table 2.
250
and casein-stimulated ATPase reactions, respectively. Despite
the inclusion of 5 µM monomeric Lon in the reactions
however, only ∼2 µM burst of ADP production was detected
in all three time courses, suggesting that only 40% of the
Lon monomer hydrolyzes ATP during the first enzyme
turnover.
Comparison of ATPase and S3 CleaVage. To assess how
ATP hydrolysis and peptide cleavage are kinetically coordinated, we compared the time courses of peptide cleavage
and peptide-stimulated ATP hydrolysis under identical reaction conditions as shown in Figure 6 (5 µM Lon monomer,
100 µM ATP, and 500 µM peptide). The pre-steady-state
phase of ATP hydrolysis consists of the burst of ADP
production (kburst,ATP ) 6.5 ( 1.4 s-1) and the transition phase,
which together spans the first 400 ms of the reaction. Steadystate turnover of ATP hydrolysis, which reflects mostly the
rate-limiting step of the reaction, occurs thereafter with a
turnover number of 0.69 ( 0.01 s-1. Under identical reaction
conditions, the lag in S3 cleavage persists for approximately
1 s with a lag rate constant of 0.94 ( 0.08 s-1 prior to the
attainment of steady-state turnover in S3 cleavage at 3.7 (
0.2 s-1. The close agreement between the lag rate constant
for peptide hydrolysis and the kcat of peptide-stimulated
ATPase suggests that the first turnover of peptide cleavage
may be coupled with the rate-limiting step of peptidestimulated ATPase activity.
As indicated in the above experiments, despite the inclusion of 5 µM Lon monomer in the ATPase reactions, only
2 µM ADP formation were detected in the burst phases of
the ATPase time course. Because the acid-quench experiments measured ADP formation at the active site of the
enzyme, the detection of a substoichiometric burst could be
attributed to 40% of the enzyme saturated with ATP because
of the low nucleotide concentration. If this explanation is
correct, the burst amplitude, which reflects the active ATPase
concentration, should increase with the nucleotide concentration. To test this hypothesis, we monitored the ATPase
reaction in the presence of 5 µM Lon monomer, 500 µM
S2, and varying [ATP] for 1.8 s. Figure 7 shows that the
time courses display triphasic behavior as discussed above
in Figure 5A. The steady-state rates of ATP hydrolysis were
obtained by fitting the data from 600 ms to 1.8 s with a
linear function. Plotting the observed steady-state rate
constants of ATPase (kss,ATP) versus the ATP concentration
yields Figure 8A, which was best fit with eq 2. The kcat,ATP
value determined from this analysis was 1.4 ( 0.1 s-1, and
the Km,ATP was 76 ( 33 µM, both of which closely agree
with that of 1 s-1 and 49 µM determined previously at 150
nM monomeric Lon (23). The Hill coefficient obtained from
the fit of these data was 1.7 ( 0.2. Although E. coli Lon
exists predominantly as a homooligomer, it exhibits at least
two different affinities for ATP (Kd < 1 µM and Kd ) 10
µM) (19). Because each monomeric subunit contains only
one ATP-binding site, it is conceivable that the ATPase sites
in oligomeric Lon are asymmetrical in their functions.
Therefore, the detection of a Hill coefficient close to 2 could
be a measurement of the communication between two
functionally nonequivalent subunits during enzyme catalysis.
This speculation however, will require further evaluation by
biophysical approaches that relate the oligomeric state of Lon
with enzymatic activities.
Kinetic Mechanism of ATP Hydrolysis and Peptide Cleavage
Biochemistry, Vol. 44, No. 5, 2005 1677
Table 2: Parameters Obtained from Pre-steady-State Kinetic Characterization of the ATPase Activity of E. coli Lon
kcat,ATP
Km,ATP
n
KATP
kburst
burst amplitude
intrinsic ATPase
+20 µM casein
+500 µM S2
NA
NA
NA
NA
7.6 ( 1.3 s-1 c
1.92 ( 0.12 µMc
NA
NA
NA
NA
12.2 ( 3.2 s-1 c
1.8 ( 0.2 µMc
1.4 ( 0.1 s-1 a
76 ( 33 µMa
1.7 ( 0.2a (1.4 ( 0.1)b
22 ( 16 µMb
6.5 ( 1.4c (11.3 ( 3.3)a (18 ( 2) sec-1 d
2.06 ( 0.15c (2.2 ( 0.1)b (4.1 ( 0.1) µMd
a
These values were obtained by fitting the data in Figure 8A with eq 2. b These values were obtained by fitting the data in Figure 8B with eq
2. c These values were obtained by fitting the acid-quenched time courses (0-400 ms) of ATP hydrolysis that are shown in Figure 5C with eq 5.
d
These values were obtained by fitting the pulse-chase data in Figure 10 with eq 5.
FIGURE 6: Pre-steady-state time courses for ATP hydrolysis and
S3 degradation at identical reaction conditions. Monomeric Lon
(5 µM) was incubated with 100 µM ATP and 500 µM peptide
substrate. The time course for peptide hydrolysis (O) was determined by fluorescence stopped-flow spectroscopy using 100 µM
nonradiolabeled ATP and 500 µM S3 as the substrates. The time
course for ATP hydrolysis ([) was determined by rapid acid
quenching of a reaction containing Lon, 100 µM [R-32P]ATP, and
500 µM S2 as described in the Materials and Methods. The values
on the left y axis were obtained by dividing the concentrations of
the peptide or ATP hydrolyzed by 5 µM Lon, whereas the right y
axis reports the concentrations of products formed.
FIGURE 7: Pre-steady-state time courses at varying [ATP] and
constant S2. Monomeric Lon (5 µM) was incubated with 500 µM
S2 and 5 (4), 10 (b), 25 (]), 50 (0), 100 (×), and 200 (O) µM
[R-32P]ATP. The reactions were rapidly quenched with acid at the
indicated times, and the amount of [R-32P]ADP formed was
determined by PhosphorImaging. Each time course was repeated
at least 3 times, and the averaged data are reported.
The burst amplitudes and burst rate constants were
obtained by fitting the data spanning 0-400 ms in Figure 7
with eq 5 and plotting the respective parameters against their
specific ATP concentration (parts B and C of Figure 8).
Because the burst amplitude directly reflects the amount of
ADP production at the active site of the enzyme, the fit of
the data in Figure 8B with eq 2 provided a measurement of
the apparent Kd for ATP (KATP). As shown in Figure 8B,
251
the burst amplitudes for ATP hydrolysis exhibit a dependency
on the ATP concentration and a maximum burst amplitude
of 2.2 ( 0.1 µM (44% of the concentration of Lon monomer
present), a KATP of 22 ( 16 µM, and a Hill coefficient of
1.4 ( 0.10 were obtained through fitting the data with eq 2.
According to Figure 8C, the burst rates of ATP hydrolysis
are independent of the nucleotide concentrations (5-200 µM)
and the averaged burst rate constant (kburst) is 11.3 ( 3.3
s-1. At 5 µM ATP, it is anticipated that the tight but not the
weak nucleotide-binding sites of Lon will be saturated with
ATP. Therefore, the apparent independence of kburst toward
the indicated ATP concentrations is likely attributed to ATP
hydrolysis occurring at the high-affinity sites. When our data
are taken together with the observed 44% burst amplitude,
they suggest that the pre-steady-state burst in ATP hydrolysis
occurs at the high-affinity sites (∼50% of the total monomer
concentration) but the hydrolysis of ATP is coordinated with
ATP binding at the low-affinity sites.
Characterization of ATP Binding. The substoichiometric
burst in ADP production could be caused by either inefficient
nucleotide binding or reflects the coordinated ATP binding
and hydrolysis between the nonequivalent ATPase sites in
Lon. To further evaluate these potential mechanisms, we
measured the concentration of functional nucleotide-binding
and hydrolysis sites using a filter binding assay and a pulsechase experiment, respectively.
In the filter binding assay, several aliquots of an enzyme
stock of Lon, whose concentration was predetermined by
the Bradford assay (final enzyme concentration e 5 µM),
were incubated with 10 µM [R-32P]ATP at 37 °C to yield
[R-32P]ADP that remained tightly bound to Lon. Because
ADP competes with ATP for the same binding site and its
Ki is 0.3 µM (11), it is anticipated that the concentration of
active Lon could be defined by the population of enzyme
that bound [R-32P]ADP and was retained as radiolabeled
protein onto the nitrocellulose. As such, the concentration
of active Lon could be determined from the radioactive
counts of standards containing known ATP concentrations.
A plot of [32P]ADP versus the volume of Lon stock added
in the binding reaction yielded a linear plot (Figure 9),
indicating that the concentration of Lon/[32P]ADP formed
was directly proportional to the amount of enzyme present
in the reaction. The concentration of active Lon was
calculated from the slope of the fit, which corresponds to
36 µM monomeric enzyme. This value agrees closely with
the concentration of the enzyme stock determined by the
Bradford assay (35 µM) and indicates that all of the
monomeric Lon used in the ATPase reactions could bind
the nucleotide.
1678 Biochemistry, Vol. 44, No. 5, 2005
Vineyard et al.
FIGURE 9: Determining the concentration of active Lon by filter
binding assay. Total of 2, 3, 4, and 5 µL of a Lon stock (35 µM
monomer as determined by the Bradford assay) was incubated with
10 µM [R-32P]ATP at 37 °C for 20 min to convert ATP to ADP.
The amount of Lon/[R-32P]ADP formed in each reaction was
determined by the filter binding assay described in the Materials
and Methods. Plotting the amount of [R-32P]ADP bound to Lon
against the volume of enzyme stock added yields a straight line
that was fit with the linear equation y ) mx + c, where m equals
36 pmol of ADP/µL of Lon (36 µM Lon momomer).
FIGURE 8: Fitting the pre-steady-state kinetic parameters of ATP
hydrolysis by Lon. (A) Data from 600 ms to 1.8 s in Figure 7 were
fit with a linear function to provide the steady-state rates of ATP
hydrolysis at varying [ATP]. Each time point was performed at
least 3 times, and the averaged data were reported. The error bars
represent the error of the fit for each data point. The kss,ATP values
were obtained by dividing the steady-state rates by [Lon]. Plotting
the kss,ATP values against its specific [ATP] yields a sigmoidal plot
that is best fit with eq 2. The kinetic parameters obtained from the
fit were kcat,ATP ) 1.4 ( 0.1 s-1, Km,ATP ) 76 ( 33 µM, and n )
1.7 ( 0.2 and are summarized in Table 2. (B) Burst amplitudes
were determined by fitting the data from 0 to 400 ms in Figure 7
with eq 5. As summarized in Table 2, the maximum burst amplitude
obtained from the fit was 2.2 µM, which corresponds to ∼44% of
the enzyme present in the reaction, a KATP ) 22 ( 16 µM, and n
) 1.4 ( 0.1. The time points reported here are averaged values of
three different trials. The error bars represent the standard error of
the fit for evaluating the respective burst amplitude values at the
specific [ATP]. (C) The burst rates of ATP hydrolysis were
determined by the same manner as in B, and the average kburst )
11.3 ( 3.3 s-1.
To determine if all of the monomeric Lon prepared for
this study could hydrolyze ATP, we performed a pulse-chase
252
experiment. In this experiment, 5 µM Lon monomer was
incubated with 500 µM S2 and rapidly mixed with 100 µM
[R-32P]ATP as in the chemical-quench-flow experiment,
except that the mixed reaction was chased with 10 mM
unlabeled Mg‚ATP for 60 s before quenching with acid and
denaturation with phenol/chloroform. During ATP hydrolysis, [R-32P]ATP bound to Lon could either be hydrolyzed to
yield [R-32P]ADP or dissociate from the enzyme. In the
presence of 10 mM unlabeled ATP (100-fold excess over
labeled ATP), any enzyme not bound to [R-32P]ATP would
be sequestered by the unlabeled ATP and not be detected in
the assay. In the acid-quench experiment, any [R-32P]ATP
that bound to Lon but was not hydrolyzed remained as
unreacted ATP. In contrast, the 60 s delay in the pulse-chase
experiment allowed time for the enzyme to hydrolyze any
[R-32P]ATP that was bound during the various incubation
times, and the presence of 10 mM unlabeled ATP (100-fold
excess over labeled ATP) allowed for the complete exchange
of [R-32P]ADP for unlabeled ATP.
Figure 10 compares the time courses of ATP hydrolysis
by Lon under the acid-quench and pulse-chase conditions.
The data spanning the first 400 ms were fit with eq 5 to
yield the burst amplitudes and kburst of the respective
reactions. In the presence of ATP chase, the burst amplitude
was 4.1 ( 0.1 µM, which was approximately 80% of the
total monomeric Lon present in the reaction. The burst rate
of the chased reaction was 18 ( 2 s-1, which was 2-fold
higher than that determined for the chemical-quench condition under identical labeled ATP conditions. The pulse-chase
result showed that at least 80% of the Lon monomer could
bind and hydrolyze ATP. Therefore, the detection of a
reduced burst in ADP production in the chemical-quenchflow time course was not due to the presence of inactive
enzyme in the reaction. Moreover, the ATPase sites of Lon
appeared to be functionally nonequivalent, because ∼50%
of the sites (tight affinity) hydrolyzed ATP within the first
400 ms of the reaction, whereas the remaining sites (weak
affinity) hydrolyzed ATP at a much slower rate.
Kinetic Mechanism of ATP Hydrolysis and Peptide Cleavage
Biochemistry, Vol. 44, No. 5, 2005 1679
Scheme
1a
a E and F are different catalytic forms of Lon along the reaction
pathway.
FIGURE 10: Monomeric Lon (5 µM) was incubated with 500 µΜ
S2 and 100 µM [R-32P]ATP for the indicated times and then
quenched with acid (O) or chased with 10 mM Mg‚ATP for 60 s
prior to quenching with acid (b). The data from 0 to 400 ms were
best-fit with eq 5 to yield a burst amplitude of 2.06 ( 0.15 µM
and a kburst of 6.2 ( 1.4 s-1 for the acid-quench experiment.
Similarly, a burst amplitude of 4.1 ( 0.1 µM and a kburst of 18 (
2 s-1 were yielded for the pulse-chase experiment. The amount of
[R-32P]ADP generated at each time point was reported on the right
y axis. The values on the left y axis were obtained by dividing the
concentrations of ADP formed by 5 µM Lon.
DISCUSSION
Lon is an ATP-dependent protease functioning to degrade
damaged and certain regulatory proteins in ViVo. In this study,
we demonstrated that in E. coli Lon, peptide cleavage is
driven by ATP hydrolysis, which is coordinated with ATP
binding to the low-affinity sites in the oligomeric enzyme.
Our findings are in discord with the previous proposal that
peptide cleavage occurs before ATP hydrolysis in Lon
catalysis (3, 4, 6, 21). In the previous studies, on the basis
that proteins but not tetrapeptide substrates stimulate the
ATPase activity of Lon, it was proposed that proteins bind
to an allosteric site in Lon to promote ADP release, thereby
facilitating enzyme turnover (3, 19). Although it is known
that ATP is the most effective activator of Lon protease,
mechanistic details concerning how ATP hydrolysis is
coupled with peptide bond cleavage is not available. To
investigate the molecular mechanism of the ATPase-dependent peptidase activity in E. coli Lon, we performed the first
pre-steady-state kinetic analysis on this enzyme by measuring
the kinetics of the first turnover of ATP and peptide
hydrolysis of a synthetic peptide substrate whose degradation
by Lon exhibits the same ATP dependency as protein
substrates (22). Because this peptide contains only one Lon
cleavage site and it stimulates ATP hydrolysis, the data
obtained from this study can be directly attributed to the
ATPase-dependent peptidase reaction rather than polypeptide
unfolding or processive peptide cleavage.
As presented in this study, E. coli Lon exhibits lag kinetics
in the degradation of the model peptide but burst kinetics in
ATP hydrolysis. During the lag in peptide cleavage, ∼50%
of the Lon monomer hydrolyzes ATP with a burst rate
constant of 11.3 ( 3.3 s-1. According to the ATPase kinetic
data, the kcat for peptide-stimulated ATPase is 1.4 s -1 (Table
2), which approximates the klag,S3 of S3 cleavage as summarized in Table 1 (0.88-1.14 s-1). These results show that
the first turnover of peptide hydrolysis requires the build up
of a reaction intermediate, which coincides with the rapid
hydrolysis of ATP during the first enzyme turnover. The
253
burst in ATP hydrolysis further indicates that the rate-limiting
step of the reaction occurs after nucleotide hydrolysis;
therefore, it could be ADP product release. The detection of
the rate-limiting step following ATP hydrolysis is consistent
with the proposal that ADP release limits ATPase turnover
(3, 19) and is supported by our observation that both casein
and S2 peptide stimulate only the steady-state turnover rate
of ATP hydrolysis (Figure 5A). Collectively, these results
support a reaction model in which activation of peptide
hydrolysis is driven by ATP hydrolysis and the first turnover
of peptide cleavage is coupled with the rate-limiting step in
ATP hydrolysis.
This model indicates that ATP hydrolysis precedes peptide
cleavage and is in discord with the earlier reaction model
proposed for Lon, which suggests that peptide hydrolysis
occurs before ATP consumption (3). The discrepancy
between the two models could be attributed to the choice of
peptide substrates used in evaluating the ATPase dependency
of the reaction. Some of the earlier studies utilized peptides
lacking ATPase stimulation abilities as substrates, which
might have led to an underestimation of the contribution of
ATP hydrolysis toward peptide-cleavage efficiency. Although
S2 is significantly smaller than casein and contains only one
Lon cleavage site, it exhibits the same ATPase stimulation
profile as the latter (23). Together with the previous data
indicating that the kcat for peptide degradation is 7-fold higher
in the presence of ATP versus AMPPNP, we conclude that
this peptide is more suitable for evaluating the ATPasedependent protease mechanism of Lon (11).
Pre-steady state kinetic analysis of the ATPase activity of
Lon further reveals functional nonequivalency in the subunits
of the enzyme, because only 50% of the ATP bound to Lon
is hydrolyzed before peptide cleavage. The observed asymmetry in the ATPase activity could be attributed to the two
different classes of ATP-binding sites found in E. coli Lon
as reported by Menon and Goldberg [Kd < 1 µM and Kd ∼
10 µM (19, 20)], who also observed that optimal protein
degradation requires occupancy of ATP at each site. The
molecular basis for such requirement, however, was not clear.
On the basis of the ATPase data obtained in this study, we
propose a sequential ATP hydrolysis reaction model that
could account for the aforementioned observations (Scheme
1). Assuming that ∼50% of the Lon subunits exhibit high
affinity for ATP, these sites will be saturated at 5 µM ATP,
which is the lowest concentration of ATP used in this study.
Under this condition, Lon binds but does not hydrolyze ATP.
As the concentration of ATP increases, the low-affinity sites
become occupied with the nucleotide, which upon full
1680 Biochemistry, Vol. 44, No. 5, 2005
Vineyard et al.
FIGURE 11: Collective fit of acid-quench ATPase data using FitSim.
Simulation of the ATPase mechanism outlined in Scheme 1 was
performed using FitSim. The resulting solid lines yielded a kburst )
8 ( 2.4 s-1 and kss,ATP ) 0.8 s-1 and were overlayed with the
experimental data from Figure 7, demonstrating consistency with
the proposed sequential mechanism.
occupancy activates ATP hydrolysis at the high-affinity sites.
Hydrolysis of ATP at the low-affinity sites occurs thereafter.
On the basis of this model, the first turnover of ATP
hydrolysis, which occurs at the high-affinity sites, is dependent on the binding of ATP to the low-affinity sites and
a 50% burst in ATP hydrolysis is anticipated in the burst
phase of the acid-quench experiment as shown in Figures
8B and 10. However, when chased with unlabeled ATP, the
radiolabeled ATP bound to the low-affinity sites will
eventually hydrolyze ATP to yield a full burst in ADP
formation (Figure 10). Because initial ATP hydrolysis occurs
at the high-affinity sites, the burst amplitudes but not the
burst rates are dependent on ATP binding to the low-affinity
sites (parts B and C of Figure 8). Because peptide hydrolysis
is coupled with a step after ATP hydrolysis, the binding of
ATP to the low-affinity sites therefore indirectly activates
the peptidase activity by stimulating ATP hydrolysis. The
triphasic ATPase time courses together with the half-site
reactivity in ATP hydrolysis are similar to the ATPase
activity of yeast topoisomerase in which its sequential
ATPase mechanism contains a burst phase and two slow
steps of comparable magnitudes (27). It is possible that Lon
adopts a similar mechanism in coupling ATPase activity to
activate peptide cleavage. To further evaluate this hypothesis,
we fit the averaged time courses of the acid-quenched
ATPase data (from 5 to 100 µM ATP) collectively to the
kinetic mechanism shown in Scheme 1 by regression analysis
using FitSim (28-30) or KinFitSim (31). This minimal
mechanism features the sequential ATPase mechanism
deduced based upon the data analyses presented above and
assumes that one of the product-release steps limits enzyme
turnover. In addition, the concentration of enzyme used for
the fitting process was varied at the respective ATP
concentration based upon the data reported in Figure 8B.
The two kon and koff for ATP binding are estimated from the
two ATP affinities of Lon [<1 and 10 µM, respectively (19)].
Because the kinetics of product release are yet to be
determined, the rate constants associated with events after
steady-state turnover are at present hypothetical. Fitting the
data to this mechanism yielded a pre-steady-state burst rate
constant for ATP hydrolysis of 8 ( 2.4 sec-1 and a steadystate turnover of 4 ( 0.34 µM/sec (Figure 11), which, upon
division by the 5 µM Lon monomer present in the reaction,
254
yielded the steady-state turnover number for ATP hydrolysis
of 0.8 sec-1. These values are in accordance with the
maximum kburst of 11.3 ( 3.3 s-1 and kcat,ATP of 1.4 ( 0.1
s-1 determined by analyzing the pre-steady-state and steadystate time course of ATP hydrolysis separately as described
above (Table 2). While the ATPase time courses presented
in this study could be consistent with a sequential reaction
mechanism of Lon, these experiments alone cannot conclusively determine this mechanism. Therefore, additional presteady-state kinetic characterizations are currently underway
to further investigate the ATPase reaction mechanism of Lon
in more detail.
The ATPase mechanism proposed in this study predicts
that communication between the two classes of ATPase sites
contribute significantly to peptide cleavage. This proposal
is supported by the observation that both the ATPase and
peptidase sites display the same level of positive cooperativity in the binding and hydrolysis of substrates. We observe
that the klag as well as the observed steady-state rate constant
(kss,S3) values of peptide cleavage exhibit sigmoidal dependency on S3 concentrations with a Hill coefficient (n) ranging
from 1.22 to 1.7 (Table 1). Moreover, both the steady-state
turnover number as well as the burst amplitude of ATP
hydrolysis determined at saturating S2 and varying ATP
concentrations also exhibit sigmoidal kinetics. Assuming that
E. coli Lon is a hexamer (14), the maximum n value would
be 6, and thus, an n value of ∼1.6 measured here either
suggests slight positive cooperativity among the six Lon
subunits or the communication between the two classes of
ATPase sites with a maximum of n ) 2. While the extent
of subunit communication cannot be defined in this study,
our results clearly reveal that interactions among the subunits
in Lon affect the ATPase and peptidase activities of the
enzyme.
Because ATP hydrolysis does occur before S3 cleavage,
we propose that nucleotide hydrolysis generates an enzyme
form that subsequently cleaves S3. Lon exhibits high
sequence and structural homology with the heterosubunit
ATP-dependent proteases, such as HslUV, which utilizes
ATP hydrolysis to deliver unfolded polypeptide substrates
to the proteolytic site (14, 15, 17, 32-34). This similarity
suggests that ATP consumption serves a similar function in
Lon. Previously, we employed steady-state kinetics and
product inhibition studies to construct a kinetic model to
account for the observed “ATPase-dependent” S3 cleavage
reaction by Lon (11). Because the identities of different
enzyme intermediates existing along the peptidase reaction
pathway are not defined, we cannot fit the time courses of
the peptidase reactions to a defined kinetic mechanism as in
the case for the ATPase reaction. However, a simplified
version of this model showing the microscopic events
associated with S3 cleavage is provided in Scheme 2. This
model proposes that ATP hydrolysis facilitates the delivery
of S3 to the peptidase site of Lon and the exchange of ADP
with ATP in step 3 is rate-limiting. In the presence of excess
S3, the peptide binds to an allosteric site in Lon to promote
the exchange of ADP with ATP, thereby increasing the
catalytic turnover of ATP hydrolysis. The data obtained in
this study support this proposed model by revealing that ATP
hydrolysis precedes peptide bond cleavage. The hydrolysis
of ATP (step 2) occurs with an apparent rate constant of
∼11 s-1 (Table 2) to generate an active enzyme form “F”
Kinetic Mechanism of ATP Hydrolysis and Peptide Cleavage
Scheme 2
that, upon the exchange of ADP with ATP, cleaves S3 (step
3). We tentatively assign step 3 as the slow step because
ADP inhibits S3 cleavage and its slow release from Lon
would be consistent with the detection of burst kinetics in
ATP hydrolysis (Figure 5). If F/ATP/S3 is the enzyme form
that catalyzes peptide bond cleavage and subsequent steps
associated with S3 cleavage are fast, then the kinetics of S3
cleavage will exhibit a lag whose duration depends on the
kinetics of ADP and/or Pi dissociation. Because the lag rate
constant for S3 cleavage is ∼1 s-1 (Table 1) and the kcat of
ATP hydrolysis is ∼1.2 s-1 (Table 2), it is conceivable that
such coordination between the two hydrolytic events occurs
in the first turnover of S3 cleavage. However, it is also
possible that the kcat of ATP hydrolysis and the klag of S3
cleavage are coordinated successively in the reaction pathway
of Lon. In this case, both steps contribute sequentially to
limit the turnover of peptide hydrolysis. The data presented
in this study cannot distinguish between these two possibilities, and further kinetic characterization is needed to further
delineate this mechanism.
In addition to peptide translocation, the ATPase activity
of Lon could also be used to facilitate other kinetic events
prior to peptide cleavage. For example, because Lon functions as an oligomer, ATP hydrolysis may be used to promote
enzyme oligomerization or to induce a conformational change
in the oligomer that facilitates peptide cleavage. The kinetic
data presented in this study cannot disprove these possibilities
because these events occur before S3 cleavage. However,
studies performed on the oligomerization of Lon homologues
(3, 35, 36) thus far have revealed that the enzyme oligomerization is independent of nucleotide, thereby excluding
the possibility that ATP consumption is coupled to enzyme
oligomerization. However, unpublished data (Lee and Burke)
suggest that the oligomerization of Lon is a reversible process
that is sensitive to reaction conditions. As such, studies are
currently being conducted to evaluate the effect of ATP on
the oligomeric state of Lon under the reaction conditions
used in these studies.
ACKNOWLEDGMENT
We thank Hilary Frase and Joyce Jentoft for their
assistance in preparing this manuscript.
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BI048618Z
7432
Biochemistry 2004, 43, 7432-7442
Correlation of an Adenine-Specific Conformational Change with the
ATP-Dependent Peptidase Activity of Escherichia coli Lon†
Jessica Patterson,‡,§ Diana Vineyard,‡,§ Jennifer Thomas-Wohlever,‡ Ramona Behshad,‡ Morris Burke,| and
Irene Lee*,‡
Department of Chemistry and Department of Biology, Case Western ReserVe UniVersity, CleVeland, Ohio 44106
ReceiVed December 2, 2003; ReVised Manuscript ReceiVed March 12, 2004
ABSTRACT: Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP
and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide
cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable
nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the kcat of peptide cleavage decreases
with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP
∼ UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of
Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion
data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The
kinetic data that were obtained were further put into the context of the structural organization of Lon
protease by probing the conformational change in Lon bound to the different nucleotides. Both adeninecontaining nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67
kDa fragment contains the ATP binding pocket (also known as the R/β domain), the substrate sensor and
discriminatory (SSD) domain (also known as the R-helical domain), and the protease domain of Lon, we
propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of
nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.
Lon, also known as protease La, is an oligomeric ATPdependent protease which functions to degrade abnormal and
certain short-lived regulatory proteins in the cell (1-9). Lon
represents one of the simplest forms of ATP-dependent
proteases because both the ATPase and the protease domain
are located within each monomeric subunit (10, 11). The
functional form of Escherichia coli Lon has been shown to
exist as a tetramer or an octamer, depending on the buffer
composition (3). Recently, the crystal structure of an inactive
mutant of the Lon protease domain has been reported. This
hexameric structure lacks the ATPase domain and contains
an active site Ser to Ala mutation. The structure of this
protein displays a central cavity, which is commonly found
in the ATPase and protease subunits of other ATP-dependent
proteases (12). In addition to the protease domain, the
structure of the R-subdomain of the ATPase subunit of Lon
has been determined. This subdomain constitutes the last 25%
of the carboxyl region of the intact ATPase domain of Lon;
however, it lacks the conserved Walker motifs found in ATP
binding proteins (12, 13). Nevertheless, a structural model
consisting of the R-subdomain and the protease domain of
†
This work was supported in part by NIH Grant GM067172 and
the PRI award sponsored by the Ohio Board of Regents. R.B. is the
recipient of the SPUR fellowship, a program sponsored by the Howard
Hughes Medical Institute.
* To whom correspondence should be addressed. Phone: (216) 3686001. Fax: (216) 368-3006. E-mail: [email protected].
‡
Department of Chemistry.
§
These authors contributed equally to the preparation of the
manuscript.
|
Department of Biology.
Lon could be superimposed on the HslUV structure (12),
thereby validating the utilization of HslU as a structural
model in studying the ATP-dependent peptidase reaction of
Lon.
The heterosubunit ATP-dependent protease HslU/HslV is
a bacterial homologue of the proteasome (14). It contains
an oligomeric ATPase subunit and an oligomeric protease
subunit which assemble to form a functional enzyme (1517). The HslU subunit is an ATPase that functions to unfold
and translocate polypeptide substrates through the central
cavities of the oligomeric enzyme by a threading mechanism
(18-20). In light of the structural similarities shared by Lon
and HslU/HslV, as well as the high degree of sequence
homology in the ATPase domains of the two enzymes (21),
it is conceivable that Lon also couples ATP hydrolysis with
substrate unfolding and translocation. Furthermore, due to
their sequence homology with many AAA+ (ATPaseassociated cellular activities) proteins, both Lon and HslU
are classified as AAA+ proteins (21). The AAA+ motif
typically contains an ATP binding site in the R/β-domain
and a substrate sensor and discriminatory (SSD) site in the
R-helical domain. It is believed that the movements of these
two domains initiate chemical energy transduction in enzyme
catalysis (21, 22).
The structural data on Lon cannot conclusively demonstrate that it possesses an ATP-dependent peptide translocation step. However, we have obtained kinetic data to
support the existence of such a mechanism. In our earlier
studies, we reported that the kcat of the degradation of a
10.1021/bi036165c CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/15/2004
Reproduced with permission from J. Patterson & D. Vineyard et al, Biochemistry 43, 7432 (2004).
Copyright 2004 American Chemical Society.
257
Conformational Change in Lon and Enzyme Activities
Biochemistry, Vol. 43, No. 23, 2004 7433
1
defined peptide (S3) by Lon is maximized by ATP hydrolysis. The defined model peptide substrate (S3) and its
nonfluorescent analogue (S2) were previously used as
simplified mimics of the E. coli Lon substrate, the lambda
N protein, to demonstrate that catalytic turnover of peptide
cleavage exhibits a dependence on ATP hydrolysis (23, 24).
Since these model peptides lack any defined secondary
structure (24), the observed ATPase dependency could reflect
a peptide translocation step (18-20, 25, 26). Using product
inhibition studies, we further demonstrated that the inhibitory
effect of ADP toward Lon could be alleviated by increasing
peptide substrate concentrations. This result allowed for the
construction of a minimal kinetic model to account for the
ATPase-coupled peptide translocation step prior to substrate
cleavage (23). According to our model, ATP hydrolysis could
be used to thread the unfolded peptide substrate through the
central cavity of the oligomeric enzyme, thereby delivering
the substrate to the proteolytic site. The nonhydrolyzable
ATP analogue, AMPPCP, cannot support peptide cleavage
because of the lack of energy transduction. Another nonhydrolyzable analogue, AMPPNP, supports peptide cleavage
with an efficiency reduced compared to that of ATP. Peptide
cleavage occurs because the structure of the AMPPNPmagnesium complex may have mimicked a transition state
in ATP hydrolysis, thus inducing a conformational change
in Lon allowing for the delivery of the peptide (27).
However, because AMPPNP is not hydrolyzed by Lon, the
substrate translocation step is less efficient due to a lack of
energy transduction.
As NTP-activated casein degradation is accompanied by
hydrolysis of the respective nucleotide (at 0.5 mM nucleotide), it is conceivable that the observed differences in the
respective NTPase-dependent reactions are attributed to the
differences in the nucleotide base structure. Therefore, under
saturating NTP conditions, Lon will exist primarily as the
Lon-NTP form, and the functional relationship between the
kcat of NTP hydrolysis and peptidase activation could be
quantitatively measured. Our proposed reaction model predicts that at saturating NTP concentrations, the hydrolyzable
nucleotides that bind with weaker affinity to Lon than ATP
will support peptide degradation with accompanying hydrolysis of the nucleotide triphosphate backbone. Furthermore, the kcat for S3 cleavage with the hydrolyzable
nucleotides should be at least comparable to or higher than
that with the nonhydrolyzable ATP analogue-mediated
reaction if energy transduction affects peptide translocation.
To test our hypothesis, we measured the steady-state kinetic
parameters for S3 cleavage in the presence of ATP, GTP,
CTP, or UTP, as well as the NTPase activity of Lon in the
absence and presence of S3. Since the kinetic parameters of
ATP- and AMPPNP-mediated S3 degradation have been
previously determined (23), results obtained from this study
1
Abbreviations: AMPPNP, adenylyl 5-imidodiphosphate; AMPPCP,
adenylyl (β,γ-methylene)diphosphonate; DTT, dithiothreitol; Abz,
anthranilamide; Bz, benzoic acid amide; NO2, nitro; Tris, 2-amino-2(hydroxymethyl)-1,3-propanediol; amp, ampicillin; KPi, potassium
phosphate; λ N (lambda N protein), lambda phage protein that allows
E. coli RNA polymerase to transcribe through termination signals in
the early operons of the phage; SE, standard error; S2, nonfluorescent
analogue of S3 that is cleaved by Lon in the same manner as S3
[YRGITCSGRQK(Bz)]; S3, mixed peptide substrate containing 10%
of the fluorescent peptide Y(NO2)RGITCSGRQK(Abz) and 90% S2;
NTP, nucleotide triphosphate.
258
could be readily compared with our previous observations.
The kinetic data that were obtained were further put into
the context of the structural organization of Lon protease
by probing the conformational change in Lon bound to
different nucleotides using limited tryptic digestion analyses.
Combining the kinetic data and the limited tryptic digestion
data allowed us to evaluate the functional role of a nucleotide-specific conformational change in Lon as well as to
quantify the functional relationship between NTP hydrolysis
and peptidase activation.
EXPERIMENTAL PROCEDURES
Materials. ATP (lot A-7699) was purchased from Sigma,
while CTP (lot 2077F), GTP (lot 9311C), and UTP (lot
6688F) were purchased from ICN. The [R-32P]ATP, [R-32P]CTP, [R-32P]GTP, and [R-32P]UTP were purchased from
Perkin-Elmer. Fmoc-protected amino acids, Boc-Abz, Fmocprotected Lys Wang resin, and HBTU were purchased from
Advanced ChemTech and NovaBiochem. HATU was purchased from PerSeptive Biosystem. Tris buffer, PEI-cellulose
TLC plates, ammonium molybdate, sodium citrate, malachite
green oxalate salt, cell culture media, IPTG, SBTI, TPCKtreated trypsin, PMSF, and chromatography media were
purchased form Fisher, Sigma, and ACROS Organic.
General Methods. Peptide synthesis and protein purification procedures were performed as described previously (23).
All enzyme concentrations were reported as Lon monomer
concentrations.
NTP-Dependent Peptidase Assays. Steady-state velocity
data were collected on a Fluoromax 3 spectrofluorimeter
(Horiba Group) as described previously (23). Assays contained 50 mM Tris-HCl (pH 8.1), 5 mM magnesium acetate,
5 mM DTT, and 125 nM E. coli Lon except in experiments
using GTP, where 200 nM E. coli Lon was used, with
varying concentrations (from 0 to 1 mM) of nucleotide (ATP,
CTP, GTP, and UTP) or peptide substrate S3 (from 50 to
500 µM). All assays were performed at least in triplicate,
and the averaged value of the rates determined for each set
of nucleotide and peptide concentrations was fit to eq 1 as
described previously (23) using the nonlinear program
EnzFitter (Biosoft).
V)
Vmax [A]n[B]
(KibK′a + K′a[B] + Kb[A]n + [A]n[B])
(1)
where V is the observed rate, Vmax is the maximal rate, A is
the peptide substrate, B is the nucleotide, Ka is the Michaelis
constant for A, Kib is the intrinsic dissociation constant for
B, and Kb is the Michaelis constant for B. The kcat value
was determined by dividing Vmax with the concentration of
the Lon monomer. The Km values for peptide hydrolysis were
calculated from the relationship log K′a ) n log Km, where
n is the Hill coefficient.
Radiolabeled NTPase Assays. Steady-state velocity data
for nucleotide hydrolysis were measured as described
elsewhere (28), and all reactions were performed at least in
triplicate. Briefly, for the NTPase measurements, each
reaction mixture (50 µL) contained 50 mM Tris-HCl (pH
8.1), 5 mM magnesium acetate, 2 mM DTT, and 150 nM
Lon monomer for ATP or UTP and 600 nM Lon monomer
for CTP or GTP. For the peptide-stimulated NTPase reac-
7434 Biochemistry, Vol. 43, No. 23, 2004
Patterson et al.
tions, 500 µM peptide substrate (S2) was added to each
reaction mixture, and the reactions were initiated by the
addition of NTP. Subsequently, 5 µL aliquots were quenched
in 10 µL of 0.5 N formic acid at seven time points (from 0
to 12 min). A 3 µL aliquot (ATPase) or 2 µL aliquot
(CTPase, GTPase, and UTPase) of the reaction was spotted
directly onto a PEI-cellulose TLC plate (10 cm × 20 cm)
and the plate developed in 0.3 M potassium phosphate buffer
(pH 3.4). Radiolabeled NDP nucleotide was then quantified
using the Packard Cyclone storage phosphor screen Phosphor
imager purchased from Perkin-Elmer Life Science. To
compensate for slight variations in spotting volume, the
concentration of the NDP product obtained at each time point
was corrected using an internal reference as shown in eq 2.
[NDP] )
(
)
NDPdlu
[NTP]
NTPdlu + NDPdlu
(2)
The kinetic parameters were determined by fitting the kobs
data with eq 3 using the nonlinear regression program
KaleidaGraph (Synergy).
kobs )
kobs,max[B]
Km + [B]
(3)
S2 peptide, and either 1 mM ATP, ADP, or AMPPNP, 2
mM CTP or GTP, or 3 mM UTP were started by the addition
of 1/50 (w/w) TPCK (N-p-tosyl-L-phenylalanine chloromethyl ketone)-treated trypsin with respect to Lon. At 15 and
30 min, a 3 µL reaction aliquot was quenched in 3 µg of
soybean trypsin inhibitor (SBTI) followed by boiling. The
quenched reactions were then resolved by 12.5% SDSPAGE analysis and visualized with Coomassie brilliant blue.
Identification of Tryptic Digestion Sites in Lon by Peptide
Sequencing. The trypsin cleavage sites in Lon were identified
by sequencing the Lon fragments generated by limited tryptic
digestion in the presence of 1 mM ATP or GTP. After 45
min, the reactions were quenched with PMSF (phenylmethanesulfonyl fluoride) followed by boiling, and then
resolved on a denaturing 4 to 15% gradient gel. The Lon
fragments were electroeluted onto a PVDF membrane and
sequenced by Edman degradation performed by the Molecular Biotechnology Core at the Lerner Research Institute of
the Cleveland Clinic (Cleveland, OH). The first five amino
acids at the amino termini of each Lon fragment were
determined and then matched against the primary amino acid
sequence of E. coli Lon [GenBank accession number P08177
(3)] to identify the respective trypsin cleavage sites.
RESULTS
where kobs is the observed rate constant, kobs,max is the maximal
rate, B is the nucleotide, and Km is the Michaelis-Menten
constant.
Malachite Green NTPase Assays. Steady-state velocity
data for ATP or CTP hydrolysis were also measured using
a modified assay to detect inorganic phosphate (Pi) release
(28, 29). Solutions containing 0.045% (w/v) malachite green
oxalate (MG) in deionized water, 4.2% (w/v) ammonium
molybdate (AM) in 4 N HCl, 2% (v/v) Triton X-100 in
deionized water, and 34% (w/v) sodium citrate‚2H2O in
deionized water were prepared. Prior to each NTPase assay,
a 3:1 mixture of MG and AM was made, stirred for at least
20 min, and filtered through filter paper. The Triton X-100
solution was then added to this MG/AM solution in the
amount of 100 µL per 5 mL of MG/AM solution. A solution
of NaHPO4 and NaH2PO4 (pH 8.1) was used for the
calibration standard. For the NTPase measurements, we used
a 210 µL reaction mixture containing 50 mM Tris-HCl (pH
8.1), 5 mM magnesium acetate, 2 mM DTT, various
concentrations of Lon protease (125 nM for ATP), and 500
µM peptide substrate (S2, the nonfluorescent analogue of
S3) for the peptide-stimulated NTPase assays. For all assays,
the NTPase reaction was initiated by the addition of the
nucleotide to the reaction mixture. At eight time points (from
0 to 15 min), a 25 µL aliquot was thoroughly mixed with
400 µL of an MG/AM/Triton X-100 solution. After 30 s, 50
µL of 34% sodium citrate was added for color development.
The absorbance was then recorded at 660 nm on a UV-vis
spectrophotometer for each time point. The amount of Pi
formed at each time point was determined by comparing the
absorbance of the sample to a Pi calibration curve. Initial
velocities were determined from plots of the amount of Pi
released versus time. The kinetic parameters were determined
by fitting the averaged rate data with eq 3.
Tryptic Digestions. Tryptic digest reactions in mixtures
containing 1.5 µM Lon, 50 mM Tris (pH 8.1), 5 mM
magnesium acetate, 2 mM DTT, with or without 800 µM
259
Steady-State Kinetic Analysis of Peptide CleaVage. Using
the fluorescent peptidase assay previously employed to
compare the kinetics of ATP- versus AMPPNP-mediated
peptide degradation (23), we determined the kinetic constants
of ATP-, CTP-, UTP-, and GTP-mediated S3 hydrolysis by
E. coli Lon. The NTP-dependent S3 hydrolysis reactions
were monitored by the increase in fluorescence, which
reflects the amount of peptide hydrolyzed over time. The
steady-state observed rate constants of S3 cleavage (kobs,S3)
were determined at varying concentrations of S3 and
saturating concentrations of the respective NTP. Under these
conditions, Lon exists predominantly in the Lon-NTP form,
and thus, the observed differences in the NTP-dependent
peptidase activities reflect the effect of nucleotide hydrolysis
rather than binding. Plotting kobs,S3 as a function of S3
concentrations yields sigmoidal plots as shown in Figure 1A.
On the other hand, plotting kobs of S3 hydrolysis at saturating
S3 and increasing NTP concentrations yields hyperbolic plots
(Figure 1B). The kobs,S3 values were also measured at varying
concentrations of NTP at several fixed peptide concentrations, and the full set of velocity data was fitted with eq 1 to
yield the kinetic constants summarized in Table 1. The
detection of sigmoidal kinetics at fixed nucleotide but varying
peptide concentrations indicates that the peptide concentration
term shown in eq 1 is associated with a Hill coefficient (23).
Since Lon functions as an oligomer, the detection of a Hill
coefficient of >1 at increasing peptide concentrations suggests that the enzyme subunits communicate with each other
during peptide cleavage, and the further binding of peptide
substrate stimulates peptide hydrolysis.
As summarized in Table 1, both the Km,S3 and n values
vary only slightly among the different NTP-mediated peptidase reactions, indicating these parameters are not affected
by nucleotide binding or hydrolysis. The Kib and Kb values
of the different nucleotides represent the intrinsic dissociation
constant and Michaelis constant of the individual nucleotide
Conformational Change in Lon and Enzyme Activities
Biochemistry, Vol. 43, No. 23, 2004 7435
FIGURE 1: Steady-state peptidase activity of Lon in the presence
of different NTPs. (A) Lon was preincubated with 25, 50, 100,
150, 200, and 500 µM S3 prior to the addition of 1 mM ATP (b),
CTP (9), and GTP ([) and 1.6 mM UTP (×). All assays were
performed in triplicate, and the averaged kobs values were plotted
against the corresponding S3 concentration. The data were best fit
with the Hill equation kobs ) (kobs,max[S]n)/([S]nKm), where kobs is
the observed steady-state rate constant, kobs,max is the maximum
observed steady-state rate constant, [S] is the concentration of the
peptide, and n is the Hill coefficient (30). (B) Lon was preincubated
with 500 µM S3 prior to addition of 5, 10, 50, 75, 150, and 250
µM and 1 mM ATP (b), 30, 75, 150, 200, 500, and 800 µM and
1 mM CTP (9), 50, 75, 150, 250, and 500 µM and 1 mM GTP
([), and 100, 200, 400, 600, and 800 µM and 1.6 mM UTP (×).
All assays were performed in triplicate, and the average kobs values
were plotted against the corresponding NTP concentration. The data
were best fit with eq 3.
Table 1: Steady-State Kinetic Parameters of NTP-Dependent
Cleavage of S3 by Lon
nucleotide
ATP
CTP
UTP
GTP
AMPPNPa
a
kcat,S3 ( SE Km,S3 ( SE
(s-1)
(M)
9.0 ( 0.5
4.2 ( 0.1
1.9 ( 0.2
1.7 ( 0.2
1.0 ( 0.1
102 ( 30
151 ( 43
99 ( 32
219 ( 43
77 ( 7
Kb ( SE
(M)
Kib ( SE
(M)
7(1
7.4 ( 2
100 ( 20
73 ( 14
350 ( 125 389 ( 112
200 ( 78 250 ( 92
NDb
10 ( 1
n
1.6
1.52
1.43
1.6
1.6
These values were obtained from ref 23. b Not determined.
(30), respectively. These values vary for the different NTPs.
According to Table 1, the Kib of each NTP approximates its
Kb value, indicating that the Michaelis constant reflects the
intrinsic binding affinity of the nucleotide for Lon. The kcat,S3
260
for the different NTPs also varies with the chemical structure
of the nucleotide that is used (Table 1). Comparing the kcat,S3
values of the peptidase reactions in Table 1 reveals the order
of the ability of different nucleotides to activate S3 cleavage: ATP > CTP > UTP ∼ GTP > AMPPNP. With the
exception of AMPPNP, the decreasing order in kcat,S3
mediated by different NTPs correlates with the decreasing
affinities of Lon for the respective nucleotides that are
represented by Kib and Kb (Table 1): ATP ∼ AMPPNP >
CTP > GTP > UTP. Although both ATP and AMPPNP
bind to Lon with comparable affinity, they exhibit different
stimulatory effects toward S3 degradation. On the other hand,
CTP, which has a weaker affinity for Lon than AMPPNP,
exhibits a 4.2-fold higher value of kcat,S3. Comparing the
structures of the nucleotide bases reveals that both ATP and
CTP share similarities in the N6 (in ATP) and N4 (in CTP)
amino groups in the pyrimidine ring. Therefore, it is likely
that the binding interaction between Lon and the amino group
in the nucleotides is responsible for activation of peptide
hydrolysis. Although both GTP and UTP are less effective
than ATP or CTP in activating S3 cleavage, the kcat values
of these two NTP-mediated S3 cleavages are at least
comparable if not slightly higher than that of AMPPNPmediated S3 cleavage (23, 24). Collectively, these data
suggest that despite the reduction in binding affinity between
Lon and CTP, GTP, or UTP, the hydrolysis of the triphosphate in these nucleotides confers a catalytic advantage over
the tight binding AMPPNP. This result indicates that the
kcat,S3 is not solely dependent on the affinity of Lon for the
nucleotide. The similarity in the Km,S3 values is consistent
with our previous report that the peptide substrate binds to
Lon independent of the nucleotide (23). This result is also
corroborated by the observation that the limited tryptic
digestion patterns of Lon are identical regardless of the
presence or absence of the S3 peptide substrate and further
indicate that binding of the S3 peptide does not induce any
detectable conformational change in Lon.
Although Lon contains an identical peptide sequence for
the ATPase site in each monomer, this oligomeric enzyme
has at least two different affinities for ATP. The Kd values
for the high- and low-affinity sites are 1 and 10 µM,
respectively (31, 32). Since the Kib and Kb for ATP obtained
in this study approximate 7 µM (Table 1), we have concluded
that the low-affinity ATP-binding site of Lon is reported in
Table 1. On the basis of this observation, we further utilized
the Kib values summarized in Table 1 to assess the relative
affinity of Lon for the different nucleotides. It should be
noted that Kib values only measure the effect of nucleotide
binding on activation of the peptidase activity; therefore, Kib
alone cannot reveal whether Lon binds to CTP, GTP, or UTP
using the high- or low-affinity nucleotide-binding site.
Steady-State Characterization of the NTPase ActiVities of
Lon. In addition to being a protease, Lon also possesses
intrinsic ATPase activity that is increased in the presence of
the peptide (24) or protein substrate (3, 33). Although Lon
can hydrolyze any of the NTPs used in this study in the
presence and absence of casein (34), the kinetic parameters
associated with these NTPases have not been reported. To
quantitatively characterize the intrinsic and peptide-stimulated
NTPase activitiy of Lon, we determined the steady-state
kinetic parameters of the respective nucleotide hydrolysis
in the absence and presence of the S2 peptide, the nonfluo-
7436 Biochemistry, Vol. 43, No. 23, 2004
Patterson et al.
FIGURE 2: Steady-state NTPase activity of Lon in the absence and presence of 800 µM S3 peptide. The kobs values for NTP hydrolysis were
determined in the absence (b, 9, 2) and presence (+) of S3 as described in Experimental Procedures. The concentrations of ATP that were
used were 25, 50, 100, 250, and 500 µM and 1 mM (A). The concentrations of CTP or GTP that were used were 25, 50, 100, 250, and 500
µM. The concentrations of UTP that were used were 50, 100, 200, and 600 µM and 1 and 2 mM.
Table 2: Steady-State Kinetic Parameters of NTP Hydrolysis by Lon
intrinsic
S2-stimulated
nucleotide
kcat,NTP ( SE
(s-1)
Km,NTP ( SE
(µM)
kcat/Km(NTP)
(×103 M-1 s-1)
kcat,NTP ( SE
(s-1)
Km,NTP ( SE
(µM)
kcat/Km(NTP)
(×103 M-1 s-1)
NTPase enhancement
ATP
CTP
UTP
GTP
0.26 ( 0.02
0.14 ( 0.02
0.50 ( 0.02
0.09 ( 0.02
47 ( 10
60 ( 10
100 ( 4
57 ( 5
5.5
2.3
5.0
1.8
1.0 ( 0.1
0.28 ( 0.02
1.1 ( 0.1
0.15 ( 0.02
49 ( 5
69 ( 20
132 ( 30
42 ( 11
20
4.1
8.3
3.6
3.8
2
2.2
1.7
rescent analogue of S3 (23). Two different discontinuous
assays were used to measure the steady-state rate constants
of NTP hydrolysis (kobs,NTP): a malachite green colorimetric
assay (28, 29) that monitors the release of inorganic
phosphate (Pi) and an R-32P-labeled ATPase assay that
measures [R-32P]ADP production (28). Plots of kobs,NTP as a
function of nucleotide concentrations in all the NTPase
experiments yielded hyperbolic plots (Figure 2). Using ATP
as a reference, we have observed that the kinetic parameters
obtained by the malachite green assay are comparable to
those obtained by the R-32P-labeled ATPase assay. Therefore,
with the exception of CTPase, we used the radiolabeled
NTPase assay to determine the kinetic parameters for NTP
hydrolysis because less reagent was required. With regard
to CTP hydrolysis, the intrinsic CTPase but not the peptide-
261
stimulated kinetic data generated by the radiolabeled nucleotide hydrolysis assay were highly scattered. Therefore, the
malachite green assay was used to determine the CTPase
kinetic parameters. The plots of kobs,NTP versus nucleotide
concentration in both the absence and presence of S2 are
shown in Figure 2. All kinetic assays were performed at least
in triplicate, and the averaged values at each time point were
reported in each plot. In all cases, the observed rate constant
data were fitted with eq 3 to yield the kinetic constants
summarized in Table 2.
As discerned in Figure 2, the hydrolysis of NTPs displays
Michaelis-Menten kinetics, which agrees with the detection
of hyperbolic plots in the dependency of kobs,S3 values as a
function of NTP concentrations (Figure 1B). Table 2 shows
that while the Km values for both the intrinsic and S2-
Conformational Change in Lon and Enzyme Activities
Biochemistry, Vol. 43, No. 23, 2004 7437
stimulated NTPase reactions are comparable, the kcat values
vary considerably. The NTPase enhancement value is the
ratio of the S2-stimulated kcat to the intrinsic kcat of the
respective NTP, and yields an assessment of the effect of
peptide substrate on NTPase stimulation (Table 2). As
summarized in Table 2, the kcat of the intrinsic NTPase
activity is in the following order: UTP > ATP > CTP >
GTP. On the other hand, the kcat of the S2-stimulated NTPase
activity is in the following order: ATP ∼ UTP > CTP >
GTP. Comparing the order of intrinsic versus peptidestimulated kcat values for the respective NTPase activities
reveals that the S2 peptide exhibits a modest stimulatory
effect on the ATPase activity (3.8; Table 2) compared to
the CTP, GTP, and UTPase activities (1.7-2.2; Table 2).
Furthermore, the kcat/Km value for ATP in the presence of
the peptide substrate is higher than those of the other NTPs,
thus indicating that ATP is the preferred activator.
Probing the Structural Changes in the AAA+ Motif of Lon.
To correlate the kinetic data given in Tables 1 and 2 with
the function(s) of the AAA+ chaperone motif in Lon (21,
22), we utilized limited tryptic digestion to probe the
conformational change of Lon in the absence and presence
of the S2 peptide as well as different NTPs. The domain
organization of E. coli Lon has been examined by comparing
the digestion pattern of the enzyme by overnight digestion
with V8 protease (35). Both ATP- and ADP-bound Lon are
more resistant to V8 digestion than unbound Lon, suggesting
that the enzyme undergoes a conformational change when
binding to these nucleotides. The functional role of the ATPor ADP-induced conformational change of Lon is not known.
To address this issue, we examined the susceptibility of Lon
to limited tryptic digestion under the reaction conditions used
in our kinetic studies. Using trypsin rather than V8 protease
as a probe, we were able to detect an adenine-specific
conformational change in Lon within 30 min of digestion.
The limited tryptic digestion patterns of Lon incubated in
the absence and presence of 800 µM S2 peptide (8Km,S3)
and with a saturating amount of nucleotides are shown in
Figure 3A. Since the NTPs are also hydrolyzed under these
reaction conditions, both the Lon-NDP and Lon-NTP
forms are anticipated to exist during the tryptic digestion
conditions. Identical Lon fragmentation patterns were detected in the absence or presence of the nucleotide when S2
was omitted (data not shown), indicating that the peptide
did not induce any conformational change in Lon that could
be detected by tryptic digestion. Figure 3A shows that Lon
is more resistant to tryptic digestion when incubated with
ATP than with the other NTPs, suggesting that the LonADP or Lon-ATP form adopts a more compact conformation. Since all the limited tryptic digestion results shown in
Figure 3A most likely reflect the resistance of the LonNDP complex to tryptic digestion, we performed a control
to further compare the stability of Lon bound to ADP or
AMPPNP and without nucleotide (Figure 3B). Since AMPPNP is not hydrolyzed by Lon and it supports S3 cleavage
(with reduced efficiency compared to ATP), it is used to
mimic the effect of ATP bound to Lon while resisting tryptic
digestion. According to Figure 3B, the fragmentation patterns
of Lon incubated with ATP, ADP, and AMPPNP are
comparable. However, in the digestion reaction of Lon
incubated with AMPPNP, a slightly higher level of the 35
kDa fragment is detected, suggesting that AMPPNP-bound
262
FIGURE 3: Limited tryptic digestion of Lon in the presence of
nucleotides. Lon was digested with a limiting amount of trypsin
and quenched at the indicated times with soybean trypsin inhibitor
(SBTI) as described in Experimental Procedures. The first lane
shows the molecular markers in kilodaltons (from top to bottom):
172, 110, 79, 62, 48, 37, 24, 19, 13, and 5.
Table 3: Identification of the Trypsin Digestion Sites in Lon
observed
molecular sequence
mass (kDa) identifieda
67
45
35
26
23
7
AIQKE and
ELGEM
ELGEM
LSGYT
MNPER and
SERIE
ADNEN
LSGYT
cleavage site
domains
included
A237/E241-K783
condition
ATPase, SSD,
peptidase
A237-R587
ATPase, SSD
L490-K783
SSD, peptidase
M1/S6-K236/K240 amino terminus
c
c
b
A588-K783
L490-R587
c
c
peptidase
SSD
b
a The first five-amino acid sequence of each Lon fragment was
identified by Edman degradation as described in Experimental Procedures. b Detected in the absence or presence of NTPs. c Detected in
the absence of adenine-containing nucleotides.
Lon might have a less compact conformation than ADPbound Lon.
When tryptic digestion was performed on Lon incubated
with ATP, ADP, AMPPNP, or CTP, two prominent fragments with apparent molecular masses of 67 and 26 kDa
(Table 3) were detected within 15 min. In contrast, nucleotide-free Lon, as well as the GTP- and UTP-incubated Lon,
was mostly digested by trypsin to yield fragments with
apparent molecular masses of 67, 45, 35, 26, and 7 kDa,
respectively (Table 3). Increasing the incubation time of the
limited tryptic digestion reactions did not significantly alter
7438 Biochemistry, Vol. 43, No. 23, 2004
Patterson et al.
FIGURE 4: Fragmentation of Lon resulting from limited tryptic
digestion. The peptide fragments generated from limited tryptic
digestion were acquired as described in Experimental Procedures.
The sizes of the Lon fragments were estimated on the basis of their
position on the SDS gel compared to the molecular mass markers.
The relative positions of the fragments compared to the intact Lon
monomer were deduced from the sequencing data given in Table
3.
the digestion patterns of Lon incubated with ATP or ADP.
However, the 67 and 45 kDa fragments of the reaction
mixtures containing GTP, CTP, or UTP were further digested
by trypsin to yield more of the 35, 23, and 7 kDa fragments
(Figure 3A).
The trypsin cleavage sites identified by peptide sequencing
experiments are summarized in Table 3, and the accessible
trypsin cleavage sites of Lon are illustrated in Figure 4. The
sizes of the tryptic digestion fragments determined by their
relative mobility on the 4 to 15% gradient gel with respect
to the molecular markers agreed well with the calculated
molecular mass of the Lon fragments based upon the
identities of the trypsin cleavage sites (Table 3). In the
presence of ATP or ADP, trypsin cleaves Lon primarily at
K236 or K240 to yield a 26 kDa fragment corresponding to
the amino terminus of Lon and a 67 kDa fragment corresponding to the AAA+ chaperone motif and the protease
domain of Lon [Figure 4 and Table 3 (3, 21, 22, 35-37)].
The AAA+ chaperone motif is further comprised of the
Walker A and B motifs of the ATPase domain and SSD
domain found in many ATPases (21, 22). When intact Lon
was treated with trypsin in the presence or absence of nonadenine nucleotide triphosphates, the 67 kDa fragment was
further degraded into smaller fragments (Figure 4 and Table
3). The 45 and 23 kDa fragments correspond to tryptic
cleavage at R587 which separates the AAA+ chaperone
domain from the peptidase domain in Lon. Tryptic cleavage
of the 67 kDa fragment at R489 gave a 35 kDa fragment
which contained the SSD domain and peptidase domain. The
35 kDa fragment was further cleaved by trypsin to separate
the SSD domain (7 kDa) and protease domain (23 kDa). No
fragment corresponding to the ATPase domain alone was
detected. This could be attributed to rapid degradation of
the ATPase domain by trypsin due to its relatively open
conformation as observed in the non-nucleotide-bound HslU
(27, 38-40).
Modeling the Nucleotide Base Binding Site in Lon Using
HslU as a Study Model. Because of the architectural
263
similarity shared by Lon and the ATPase HslU (12), we have
constructed a model for the nucleotide-binding site of Lon
based upon the crystal structure of HslU bound to dADP
(PDB entry 1HT2). We utilized the Swiss PDB viewer
program to construct the nucleotide binding site for one of
the monomers in HslU (the E chain) bound to dADP. This
region contains Ile17-Ile66 of the E chain, which encompasses the conserved Walker A motif found in the AAA+
protein family (Figure 5A,B). Sequence alignment of E. coli
Lon with the nucleotide binding site of HslU reveals that
the residues in the Walker A motif of Lon and HslU are
highly homologous (Figure 5C). This level of sequence
homology validates the fitting of the Lon primary amino acid
sequences flanking Ala316-Leu365 to the structure of HslU
(consisting of Ile17-Ile66) to yield a model for the nucleotide binding site in Lon (Figure 5C,D). Figure 5B depicts
the crystal structure of the region in HslU that interacts with
the N6 amino group in adenine, whereas Figure 5D illustrates
a model (based upon the sequence alignment shown in Figure
5C) showing that Lon appears to interact with the N6 amino
group in ADP via the same mechanism as HslU. In HslU,
the N6 amino group functions as a hydrogen bond donor
that could simultaneously interact with the carbonyl oxygen
in Ile18 and Val61 along the amide backbone of the enzyme.
According to Figure 5C, Val61 is located within the Walker
A motif and is conserved in Lon. Therefore, it is conceivable
that the carbonyl oxygen of Val360 in Lon makes the same
contact with the N6 amino group in adenine. Through
sequence alignment, we have also identified that the carbonyl
oxygen in the amide backbone of Gln317 in Lon (Figure
5D) could adopt the same function as Ile18 in HslU (Figure
5B). Taken together, our model indicates that Lon binds to
the adenine nucleotide via two hydrogen bond interactions
with the N6 amino group in adenine, and disruption of these
interactions may affect the affinity of Lon for the nucleotide.
DISCUSSION
Lon is one of the simplest ATP-dependent proteases within
the AAA+ protease family (3, 21, 22). On the basis of
sequence and structural similarities shared by Lon and other
ATP-dependent proteases, it is proposed that Lon also
couples ATP binding and hydrolysis to unfold and translocate
peptide substrates into its protease chamber. The unfolded
polypeptide is threaded through the central protease chamber
which is formed by oligomerization of the enzyme subunits.
In previous studies, we reported that Lon exhibited ATPase
dependency in hydrolyzing an unstructured peptide containing one Lon cleavage site (24). Through kinetic characterization of the ATP- and AMPPNP-dependent peptidase reactions, we assigned the observed ATPase dependency to an
energy-coupled peptide translocation step similar to ones
found in other ATP-dependent proteases (23). To further test
this hypothesis, we compared the steady-state kinetic parameters of NTP-dependent S3 cleavage and NTP hydrolysis
to evaluate the functional relationship between nucleotide
binding and hydrolysis with peptide cleavage. Since CTP,
GTP, and UTP also activate the proteolytic activity of Lon
and are hydrolyzed during the reaction (34), they were used
as probes to study the mechanism by which Lon transduces
chemical energy generated from ATP hydrolysis into the
movement of the peptide substrate into the protease chamber.
At saturating nucleotide concentrations, the predominant
Conformational Change in Lon and Enzyme Activities
Biochemistry, Vol. 43, No. 23, 2004 7439
FIGURE 5: Modeling the adenine binding site of Lon based upon the structure of HslU bound to ADP (PDB entry 1HT2). (A) The structure
of a monomeric HslU containing residues 16-66 is shown in blue. The structure of ADP is shown bound to the top of the cleft. (B) The
N6 amino group in adenine functions as a hydrogen bond donor that can interact with the carbonyl oxygen of Ile18 and Val61 in HslU. (C)
The sequences corresponding to the Walker A motif of Lon and HslU are highly conserved. This region was used as a reference point to
align Ala316-Leu365 of Lon with Ile17-Ile66 of HslU. The residues that are anticipated to interact with the N6 amino group of adenine
are highlighted in green and yellow. (D) On the basis of the sequence alignment, the carbonyl oxygens of Val360 and Gln317 in Lon are
proposed to form hydrogen bonds with the N6 amino group in adenine.
enzyme form is the Lon-NTP form, and thus, the kinetic
parameters associated with S3 cleavage should be related to
the effect of nucleotide binding and/or hydrolysis. In the
absence of detailed structural information revealing the
different functional states of Lon, we utilized steady-state
enzyme kinetic techniques to demonstrate that at least one
enzyme form of Lon functions to transduce the chemical
energy liberated from ATP hydrolysis to stimulate the
catalytic turnover of peptide cleavage. This provides evidence
for the existence of a peptide translocation step in Lon.
The kinetic parameters summarized in Tables 1 and 2
collectively indicate that the kcat of peptide hydrolysis is
partially coupled with energy transduction, as ATP, being
the most effective peptidase activator, is the most sensitive
to peptide-stimulated hydrolysis. As seen in Table 1, the kcat
of S3 cleavage is 9 and 4.2 s-1 for ATP and CTP,
respectively, whereas for GTP and UTP, the kcat of S3
cleavage is within the range of 1.7-1.9 s-1. Despite the
variation in kcat,S3 found among the different NTPs, the kcat,S3
values obtained for these nucleotides are higher than that
obtained for AMPPNP (Table 1) (23). Since AMPPNP is a
nonhydrolyzable analogue of ATP, it is used to evaluate the
functional role of ATP binding in Lon catalysis. Comparing
the relative affinity of ATP with that of AMPPNP in Table
1 reveals that Lon binds to both adenine nucleotides with
comparable affinities. The Kib of AMPPNP for S3 cleavage
264
is 10 µM, which is similar to the Kib for ATP (7.4 µM; Table
1) but is lower than those obtained for CTP, GTP, and UTP
(73, 250, and 389 µM, respectively; Table 1). The limited
tryptic digestion patterns of ATP- and AMPPNP-protected
Lon also show that both nucleotides induce a more compact
conformation in the enzyme than CTP, GTP, and UTP
(Figure 3A,B). Collectively, these data indicate that while
AMPPNP is not hydrolyzed by Lon, it still binds to Lon in
a manner similar to that of ATP. Since AMPPNP induces
the same conformational change in Lon as ATP or ADP, as
judged by the limited tryptic digestion study (Figure 3B),
the lower kcat for the AMPPNP-mediated S3 hydrolysis
reaction is most likely due to the lack of energy transduction
associated with nucleotide hydrolysis rather than the absence
of an adenine-specific conformational change. This result,
in conjunction with our previous observation that AMPPCP
failed to support peptide cleavage (24), provides quantitative
evidence of the fact that at least one enzyme form of Lon
couples NTP hydrolysis with peptide cleavage.
Although all the NTPs generate a kcat value for S3 cleavage
that is higher than that of the nonhydrolyzable analogue
AMPPNP, the kcat,S3 values for the NTPs differ from one
another. This difference in kcat,S3 loosely follows the ranking
of the relative affinities of Lon for the nucleotides (Kib and
Kb; Table 1), the magnitude of NTPase enhancement (Table
2), and the effectiveness of the nucleotide in protecting Lon
7440 Biochemistry, Vol. 43, No. 23, 2004
Patterson et al.
FIGURE 6: Comparison of the structures of adenine and cytidine.
An overlaid view of the structures of adenine and cytidine reveals
that the amino groups in the two nucleotide bases are in the spatial
proximity of one another. Adenine is shown in blue and cytidine
in red.
from tryptic digestion (Figure 3A). The order of peptidase
activation is as follows: ATP > CTP > GTP > UTP. This
order parallels the ability of the respective NTPs to protect
the 67 kDa fragment of Lon from tryptic digestion: ATP >
CTP > GTP ∼ UTP. According to Table 1, peptide
hydrolysis by Lon is more efficient in the presence of ATP
and CTP, which taken together with the limited tryptic
digestion data, suggests that there is an enzyme form in Lon
that exhibits higher selectivity in transducing the energy
liberated from ATP hydrolysis into the activation of peptidase
activity.
To explain why CTP is a better peptidase activator than
GTP and UTP, we constructed a structural model for the
nucleotide base binding site of Lon based upon the crystal
structure of HslU bound to dADP [Figure 5 (27, 39, 40)].
Figure 5D shows that the N6 amino group in adenine serves
as a hydrogen bond donor that could potentially interact with
the carbonyl oxygen of Gln317 and Val360 in Lon. According to this model, nucleotides capable of forming similar
contacts with Lon should mimic the functions of the N6
amino group in ATP, which in this case is the induction of
a closed enzyme form compared to unbound Lon. Figure 6
overlays the structure of adenosine and cytosine, which
shows that both amino groups are in the spatial proximity
of one another. Constrained energy minimization of ATP
and CTP performed by Pate et al. showed that the two amino
groups were 0.7 Å apart (41). Given the spatial similarity of
the two amino groups in the respective nucleotides, it is
conceivable that CTP could make contacts with Lon similar
to those of adenine and induce a relatively closed conformational change in Lon compared to GTP and UTP. CTP,
however, is not a perfect mimic of ATP, as its kcat,S3 is 2-fold
lower than that obtained for ATP and it is less effective than
any of the adenine nucleotides at protecting Lon from tryptic
digestion. The increase in Kb and Kib for CTP (Table 1) is
probably caused by the loss of interaction between Lon and
other parts of the heterocyclic base in the nucleotide. These
results suggest that the kcat,S3 value is related to the compactness of the nucleotide-induced conformation in the enzyme.
CTP, GTP, and UTP have lower kcat,S3 values than ATP
because they induce a less compact conformation in Lon,
which reduces the effectiveness of the energy transduction
265
process. Collectively, these data indicate a functional relationship between peptidase activation, enhanced peptidestimulated ATPase activities, and the nucleotide-induced
conformational change in the enzyme. The effective communication among these three factors is needed to optimize
the peptidase activity of Lon.
Using limited tryptic digestion studies, we have demonstrated that a compact structure forms between the three
functional domains of Lon, the R/β ATPase domain, the SSD
domain (also known as the R-helical domain), and the
peptidase domain, in the presence of ATP, ADP, or AMPPNP, that resists tryptic digestion. These results agree well
with the report on the domain organization of nucleotidebound Lon (35) and HslU/HslV (27). In addition to binding
the polypeptide substrate, the SSD domain has been proposed
to participate in ATP hydrolysis (21, 22, 37). The observed
2-fold higher peptide-stimulated ATPase enhancement (3.8;
Table 2) compared to the other peptide-stimulated NTPase
enhancement (∼2; Table 2) could therefore be attributed to
the weakened interaction between the SSD and the γ-phosphate of the NTP (22, 37). Although our tryptic digestion
results show that adenine nucleotides induce a closed
conformation in Lon, the adenine-specific conformational
change alone cannot support peptide cleavage. For example,
AMPPNP is bound to Lon with an affinity comparable to
that of ATP and induces a compact conformation. The kcat,S3
of AMPPNP, however, is lower than the values obtained for
the hydrolyzable nucleotides that bind to Lon with lower
affinities (Table 1), which suggests that nucleotide hydrolysis
also plays a role in activating peptide hydrolysis. This
argument is further supported by the tryptic digestion results
showing that ADP induces a compact conformation in Lon
that is slightly distinguishable from the AMPPNP-bound
enzyme form (Figure 3B).
Using the structural changes associated with binding of
HslU to an adenine nucleotide as a template, we incorporated
the data obtained in this and previous studies to discuss the
role of ATP binding and hydrolysis in mediating energy
transduction and peptide hydrolysis in Lon. This model
features an ATPase-dependent peptide translocation and
protease mechanism similar to that proposed for HslUV. The
binding and hydrolysis of ATP induce a series of conformational changes in the enzyme that render peptide substrate
access to the central protease cavity. Figure 7 depicts a
simplified model of oligomeric Lon containing only two of
the subunits which face one another to form a central cavity.
It should be noted that the other monomers are omitted for
clarity. Furthermore, our current model assumes that each
monomeric subunit is functional as an ATP-dependent
protease, and the interaction among the subunits may invoke
positive cooperativity in enzyme catalysis that is characterized by a Hill coefficient of >1. However, further experiments should be conducted to further evaluate this cooperativity.
In the absence of nucleotide, Lon adopts a relatively loose
conformation in which the ATPase, SSD, and peptidase
domains are susceptible to tryptic digestion and yield the
fragments shown in Table 3 (I in Figure 7). These loose
conformations among the subunits form a narrow opening
to the central cavity in Lon, thereby hindering the access of
the peptide substrate to the protease chamber. The binding
of ATP to the AAA+ motif in Lon induces a more compact
Conformational Change in Lon and Enzyme Activities
Biochemistry, Vol. 43, No. 23, 2004 7441
FIGURE 7: Model proposed for the different enzyme forms in Lon that couple ATP binding and hydrolysis in activating peptide hydrolysis.
This model is proposed on the basis of the structural similarities shared by Lon and HslU. An ATPase-dependent peptide translocation is
proposed in this model (see the Discussion). The R/β-subdomain and the R-helical subdomain of the AAA+ motif are in blue and green,
respectively. The protease domain is in white. The protein domains and subdomains are connected by flexible linkers.
conformation within the enzyme, which is more resistant to
tryptic digestion than form I (II in Figure 7). This structural
rearrangement opens the pore leading into the central cavity,
which renders the peptide access to the protease chamber
and facilitates the transduction of chemical energy generated
from ATP hydrolysis to the active site of the protease where
peptide cleavage occurs (form III in Figure 7). Although this
step is speculative in Lon, the coupling of ATP binding and
hydrolysis to energy transduction through conformational
changes in enzymes has been reported in molecular motors
such as myosin (42) and HslUV (27). In our studies, the
existence of this step is supported by the observed correlation
between peptide-stimulated NTPase activity and the higher
kcat,S3 obtained for the hydrolyzable nucleotides compared
to those of nonhydrolyzable ATP analogues. The mechanism
by which Lon returns to the free enzyme form is not clear.
However, on the basis of the detection of a noncompetitive
inhibition pattern for one of the hydrolyzed peptide products
against the S3 substrate, we propose that Lon isomerizes to
another form during peptide cleavage (23). Since the
hydrolyzed peptide product is a truncated version of the S3
substrate, it is anticipated to act as a competitive inhibitor
against S3. The noncompetitive inhibition pattern seems to
suggest that Lon adopts a different form upon peptide
cleavage and the postcatalytic enzyme form is specific only
for the hydrolyzed peptide product but not for S3. This
enzyme isomerization step could account for the lack of
additional S3 degradation in form IV (Figure 7).
The model proposed in Figure 7 predicts that ATP
hydrolysis should occur before peptide cleavage, and adenine
nucleotides lacking hydrogen donating properties at the C6
266
position should be poor activators of Lon. The former
prediction could be readily tested by comparing the presteady-state kinetics of peptide and ATP hydrolysis. One
anticipates that the rate constant for ATP hydrolysis will be
higher than that obtained for peptide cleavage. This endeavor
is currently being examined in our laboratory using the same
defined peptide substrate. Elucidating the timing of ATP
binding and hydrolysis should expand our mechanistic
understanding of Lon. With regard to evaluating the functional role of the hydrogen bond interaction between Lon
and the nucleotide base in adenine, we propose a detailed
investigation utilizing non-natural nucleotides. These nucleotides lacking any hydrogen bond donating properties in the
heterocyclic bases will be used as probes to evaluate the
functional role of nucleotide binding in Lon. The design and
synthesis of a series of non-natural nucleotides is currently
underway to further address this issue.
ACKNOWLEDGMENT
We thank Dr. Anthony Berdis, Hilary Frase, and Jonathon
Ipsaro for their assistance in the preparation of the manuscript.
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