Biochemistry1988,27, 1581-1587
r5 8 l
Reconstructionby Site-DirectedMutagenesisof the Transition State for the
Activation of Tyrosine by the Tyrosyl-tRNA Synthetase: A Mobile Loop
Envelopesthe Transition State in an Induced-Fit Mechanismt
Alan R. Fersht,*'Ï Jack W. Knill-Jones,THugues Bedouelle,$
and Greg Winter$
Departmentof Chemistry,Imperial Collegeof Scienceand Technology,LondonSW7 2AY, (J.K.,and MRC Laboratoryof
Molecular Biology, MRC Centre,Hills Road, CambridgeCB2 2QH, U.K.
Receiued
Junei,5, 1987;Reuised
ManuscriptReceiued
OctoberI,;,987
ABsrRAcr: Site-directedmutagenesisof the tyrosyl-tRNA synthetasefollowed by kinetic studieshas shown
that residueswhich are distant from the active site of the free enzyme are brought into play as the structure
of the enzyme changesduring catalysis. Positively charged side chains which are in mobile loops of the
enzymeenvelopethe negatively charged pyrophosphatemoiety during the transition state for the formation
of tyrosyl adenylatein an induced-fit mechanism. ResiduesLys-82 and A19-86, which are on one side of
the rim of the binding site pocket, and Lys-230 and Lys-233, which are on the other side,have been mutated
to alanine residuesand also to asparagineor glutamine. The resultant mutants still form 1 mol of tyrosyl
adenylate/mol of dimer but with rate constantsup to 8000 times lower. Constructionof differenceenergy
diagramsrevealsthat all the residuesspecificallyinteract with the transition state for the reactionand with
pyrophosphatein the E.Tyr-AMP.PP1complex. Yet, the e-NH3+groupsof Lys-230 and Lys-233 in the
crystalline enzymeare at least 8 A too lar away to interact with the pyrophosphatemoiety in the transition
state at the same time as do Lys-82 and A19-86. Binding of substratesmust, therefore, induce a conformational changein the enzymethat brings theseresiduesinto range. Consistentwith this proposalis the
observationthat all four residuesare in flexible regionsof the protein. The induced-fit mechanismallows
accessof substratesto the active site and enablesthe transition state to be completely surroundedby groups
on the protein which would otherwise block entry.
Ihe aminoacyl-tRNA synthetases
catalyzethe formation of
aminoacyl-tRNA by frrst forming an enzyme-boundaminoacyl
adenylatecomplex(eq l, =activation)and then transferring
the aminoacid to tRNA (eq2). The mechanismof activation
E + AA + ATP = E.AA-AMP + ppi
(1)
E.AA-AMP + tRNA = AA-IRNA + E + AMP (2)
has proved an enigma. Despiteintensivework on the 20
different typesof enzyme,there is no convincingevidencefrom
classicalkinetic experimentsor protein chemistry for any
uniquelyimportant residuesas usedin conventionalacid-base
or covalentcatalysis. Further, there is little convincingsequencehomologybetweensynthetases
apart from the motif
(numberedaccordingto the tyroCys-35...His-45...His-48
syl-tRNA synthetase)noted first at the active site of the
tyrosyl-and methionyl-tRNA synthetases
(Barker & Winter,
1982). The initial crystal structuresalso failed to identify
catalytic groups,but systematicsite-directedmutagenesishas
begun to unravel the catalytic mechanism(Winter et al.,
1982). Residuesthat appearto interact with the substrates
in the crystalstructureof the enzyme-bound
tyrosyl adenylate
complexhavebeenmutated. Residuesthat are nominally in
the site for binding adenosinehavebeenfound to bind ATP
not in the ground state (enzyme-tyrosine-ATP or enzymeATP complexes)but in the transitionstateand enzyme-bound
tyrosyladenylatecomplex(e.g.,Cys-35and His-48;Wells &
Fersht,1986;Fershtet al., 1986a). Mutagenesis
of His-45
indicatedthat it was involvedin binding the transitionstate
of the reagents. Subsequentmodel building of the enzymelThis work was supported by the MRC of the U.K.
I Imperial College of Scienceand Technology.
s MRC Centre.
transition-statecomplexrevealeda binding site for the 7phosphateof ATP betweenThr-40 and His-45in the transition
state, which was confirmed by mutagenesisof Thr-40
(Leatherbarrowet al., 1985;Leatherbarrow& Fersht,1987).
An important strategyfor analyzingthe resultsof mutation
hasbeento determinethe apparentcontributionto the binding
energyof the mutatedsidechainat eachstateof the reaction
from differenceenergydiagrams(Wells & Fersht,1986;Ho
& Fersht,1986). Theseare constructed
by comparison
of the
free energyprofilesof the reactionof wild-type and mutant
enzymesas determinedby measuringall the necessaryrate
and equilibriumconstantsin eq 3. Differenceenergydiagrams
TYt.-_
L.
.-
_4JP.__
h,.t yr ;-
Kt'K^'k_,
k3
E.Tvr.ATP ;E.Tyr-AMP.PPi
t.trr-AMP
' *
PPi
(3)
showdirectly the effectsof the mutation on the binding of each
substrate,intermediate,and transitionstate. A spuriouseffect
of a residuein the ATP site affecting the binding of tyrosine,
for example,is immediatelyobvious. The combinationof
sensibledifferenceenergydiagrams,sensitivekinetictests[e.g.,
the double-mutant
test (Carteret al., 1984)],but especially
the finding of linear free energyrelationships(Fersht et al.,
1986b,1987)and X-ray crystallographic
evidence(Brown et
al., 1987)providesstrongevidencethat the resultsof these
mutationsreflectjust the local effectsof the changeof side
chain rather than any grosschangein enzymestructure.
BeyondDirect CrystallographicInformation. The kinetic
methodshavebecomeevenmore importantat the next stage
of the investigationbecausewe havenow reachedthe limits
of informationpresentlyavailablefrom proteincrystallography
on direct interactionsbetweenthe enzymeand its substrates.
0006-2960
@ 1988AmericanChemicalSociety
18810427-1s81$01.s0l0
1582 BIocHEMISTRY
The crystal structuresof the free enzyme and of the enzyme-tyrosinecomplexesare thoseof a symmetricaldimer
(Brick & Blow, 1987). But, it is knownfrom solutionstudies
that only I mol of tyrosineis boundper moleof dimer (Fersht,
1975) and that binding is accompaniedby a changein the
tryptophanfluorescence.Only 1 mol of tyrosyl adenylateis
by a further similar change
formed,and this is accompanied
in fluorescence(Fersht et al., I975). Substrate-induced
conformationalchangesare clearlyindicated,but their magnitude in structural terms is unknown. But, could these
changesbring residuesdistantfrom the activesitein the crystal
structureinto the mechanism?Cluescame from a study in
which most of the basic residuesof the enzymehad been
mutated to map out the tRNA binding site (Bedouelle&
Winter, 1986). This revealedthat severalmutants (Lys Asn-233)
Asn-82,Arg - Gln-86,Lys - Asn-230,and Lys----weredefectivein activationof tyrosine. Lys-82 and A19-86
areat one sideof the entranceto the substratebinding pocket
and Lys-230and Lys-233 are at the oppositeside. We have
now characterizedthesemutantsand a further set in which
residues82, 86, 230, and 233 have each been mutated to
alanine.
PRocnnuRes
ExpBRrl{eNTAL
Materials
Restrictionand other enzymeswere obtainedfrom Boehringer-Mannheim,chemicalsfrom Sigma (London),and radiochemicalsfrom AmershamInternational.
Methods
Productionof Mutants. Mutants Lys - Asn-82,Arg Gln-86,Lys * Asn-230,and Lys - Asn-233werepreviously
constructed(Bedouelle& Winter, 1986)on a derivativeof the
originalMl3mp93TyrS vector(Winter et al., 1982)altered
in two waysin the TyrS gene: (i) a deletionof 900 bp to the
5'side of the promoter;(ii) deletionof a prematureterminator
betweenthe promoterand the start of translation[elements
2 and3 of Waye and Winter (1986)1.This improvesthe yield
of TyrTS about 3-fold in infectedcells. For experimental
detailsand the strain constructionsusedin the site-directed
seeCarter et al. (1985): the mutagenicoligomutagenesis,
(Bedouelle& Winter, 1986)wereusedin *double
nucleotides
priming' with the strand selection primer SELl on
Ml3mpg3amIVTyrTS geneIV, transfectingthe heteroduplex
into competentEscherichia coli HB2154 with a lawn of
H82151. In the presentstudy,mutant Lys * Ala-233was
constructedwith the primer 5'-GCCGCTTTCCGTTG"C*CCCGAATTTCGT-3' without strandselection,transfecting
the heteroduplexinto competentBMHTI-lSmutL with a lawn
of BMH7l-18. MutantsLys * Ala-82,Arg----Ala-86,Lys
- Ala-230, and Glu - Ala-235wereconstructedwith primers
5'-GCTTTTTG*C*CCCGCTC-3" 5'-TCAGCGTGG*C*CTCGCTTT-3" 5'-CCCGAATG*C*CGTGCCG-3" ANd
5'-GCCGCTTG*CCGTTT-3' in double priming with the
strandselectionprimer SEL3 on Ml38l9TyrTS template.
Heteroduplexwas transfectedinto competentHB2155 with
of the TyrTS gene
a lawn of HB215l. The codingsequence
(Winter et al., 1983)wascheckedwith a family of sequencing
primers(Wilkinsonet al., 1984). An asteriskfollowsa mismatchedbasein the mutagenicprimers.
Purification of Enzymes. Mutant enzymeswere expressed
in E. coli TG2 hostsfrecA form of TGl (Gibson,1984)]and
purified to electrophoretichomogeneityby modificationof
(Wilkinsonet al., 1983). An overnight
existingprocedures
cultureof E. coli TG2 in 2xTY medium(16 g of tryptone,
10 g of yeastextract,and 5 g of NaCl per liter) wasdiluted
FERSHT
ET AL.
100-foldinto 5 mL of fresh medium and grown to early log
phase(Asso- 0.05) beforeinfectingwith recombinantM13
at high multiplicityof infection()100). Growth wascontinued
at 3'7"C for t h beforebeing transferredto 500 mL of fresh,
prewarmedmedium and shakenovernight. The cells were
harvestedby centrifugationat 7000 rpm for l5 min, resuspendedin a buffer containing50 mM tris(hydroxymethyl)hydrochloride(Tris-HCl) (pH 7.8), 1 mM
aminomethane
ethyl enedi ami netetraacetiaci
c d (E D TA ), 5 m M 2fluoride,
and 0.1 mM phenylmethanesulfonyl
mercaptoethanol,
and immediatelylysedby sonication.The lysatewas heated
enzymesfrom
at 56 oC for 40 min to precipitateendogenous
E. coli and clarified by centrifugationat 15000 rpm for 30
min. The protein precipitatingat J\Vo sattrating ammonium
sul fate(0' C ) w asdi al yzedfor 1.5-3hat 4 oC aga inst50
mM potassiumphosphate(pH 6.5), 1 mM EDTA, 0.1 mM
fluoride, and 0.1 mM tetrasodium
phenylmethanesulfonyl
tyrosyl adeny(to removeany enzyme-bound
pyrophosphate
late). After further dialysisfor 1.5-3 h againstthe samebuffer
the proteinwasappliedto
minustetrasodiumpyrophosphate,
a 2 x 5 cm columncontainingDEAE-TrisacrylM equilibrated
in the samebuffer. The columnwaswashedwith the starting
buffer and elutedwith a gradientof 50-350 mM potassium
phosphate(pH 6.5) containingI mM EDTA and 0.1 mM
fluoride. The enzyme,which eluted
phenylmethanesulfonyl
at about 150mM salt,wasdialyzedagainst20 mM Tris-HCl,
fluoride
I mM EDTA, and 0.1 mM phenylmethanesulfonyl
beforepurificationon a PharmaciaFPLC Mono Q column
(Low eet al ., 1985).
Kinetic Procedures
All experimentswere performedat 25 oC in a standard
buffer containing144 mM Tris-HCl (pH 7.78), 0.14 mM
fluoride,and 10 mM MgClr. (ATP
phenylmethanesulfonyl
was addedas the magnesiumsalt to maintain the free Mg'*
at 10mM.)
Actiuation The rate constantsfor the formation of enzyme-boundtyrosyl adenylateby all the mutantsother than
Glu - Ala-235weresufficientlylow that the reactioncould
be monitoredby manualsamplingand trappingE'Tyr-AMP
disks. A solutionof V4 mM Tris-Hcl-l0
on nitrocellulose
mM MgCl2containing[taC]Tyr (210or 522 mCi/mmol) and
(0.005unit/ml) of total
yeastinorganicpyrophosphatase
volume150pL wasincubatedat 25 "C; l5-30 p"Lof enzyme
(2-10 pM) in the samebuffer was addedto initiate the reaction. Experimentsto determinethe valuesof Knafor ATP
were generallyconductedat 40 pM tyrosineand those for
determiningthe Ky for tyrosineat l0 mM ATP. Samples
(20 pL) were periodicallywithdrawn and layeredonto a nidisk which wasundermild suction. The disk was
trocellulose
then washedwith 5 mL of Tris-HCl-MgClr buffer, dried
in scintillant,and the
underan infraredlamp, and suspended
retainedE.ItaC]Tyr-AMP wasassayedby scintillationcounting. The reactionsfollowedfirst-orderkinetics,and seven
sampleswere taken over five half-lives. The reactionswere
followedto completion()10 half-lives)to obtain end points.
Pyrophosphorolysis.A solutionof enzyme-bound[1oC]Tyr-AMP wasobtainedby incubatingenzymein a buffer at
pH 6 [130 mM [bis(2-hydroxyethyl)amino]tris(hydroxy(Bis-Tris),10 mM MgCl2l in the presence
methyl)methane
of 25 pM [taC]Tyrand 25 mM Mg-ATP. The E'Tyr-AMP
complexwasdesaltedon a SephadexG-50 (medium)column
(l x 25 cm) with standardpH 7.78buffer. The enzyme-bound
werestoredunderliquid nitrogen. The
aminoacyladenylates
weresufficientlylow that
rate constantsfor pyrophosphorolysis
the reactionscouldbe monitoredby manualsamplingas above.
TY.ROSYL-TRNA
SYNTHETASE
MECHANISM
voL.
27, NO. 5, 1988
1583
Table I: Activation of Tyrosine by Tyrosyl-tRNA Synthetaseo
K, (rrM)
frr (s-t)
ktlK'^ (s-' 14-t;
&-r (s-t)
Koo (mM)
k-tl Ko, (s-t Y-t;
wild type
t2
4.7
38
8080
16.6
0.61
27 200
-40b
-2b
Lys -* Ala-82
l3
50
730
Lys - Asn-82
l3
5.2
0.3
58
0.26
1.5
170
Arg - Ala-86
7.5
9.9
5 . 0x l 0 - 3
0.51
1.2
Arg - Gln-86
4
3.5
4 . 2x l0-3
1.2
3.5 x l0-3
2.5
1.4
Lys Ala-230
23
4.9
0.39
80
130
Lys - Asn-230
l9
3.4
2 . 6 x l0-2
7.6
l5
Lys - Ala-233
10.4
3.0
9.8
Lys - Asn-233
t2
0.2
0.38
oAll experiments
conductcdin the standardbuffer at 25 oC and pH 7.78. Kr, the dissociâtion
constantofthe E.Tyr complex,wasdeterminedby
equilibrium dialysis. X'., the dissociation constânt of ATP from the E.ATP complex, k, and /ca, the rate constants for the forward and reverse
reaction, and Ko, the dissociationconstant of PPi from the E Tyr-AMP.PP; complex, were determined from pre-steady-statekinetics. tK, for some
mutantswasalsodeterminedkineticallyfrom the Éte of activationat subsaturating
conconcentrations
of ATP, and the valueswere found to be in
excellentagreementwith thoselrom equilibriumdialysis. The blank entriesin th€ tableoccur wherethe valuerof (/â and KD are too high (>50 M
and >.10mM, respectively)
to be determined.rt3and *,3 are consequently
inac.essible,
bur the ratioskj/('! and i-r/KDe may iiill be determinedwith
precrsron,
enzyme
rK'" (mM)
The reaction was initiated by the addition of tetrasodium
pyrophosphate
to a solutionof 100-400nM E.[taC]Tyr-AMP
in the standardbuffer (both solutionshaving beenincubated
at 25 oC) to give a final volume of 130 pL.
PyrophosphateExchangeKinelics. Thesewere performed
for the mutant Glu * Ala-235 in the standardbuffer (Wilkinsonet al., 1983).
Binding of Tyrosine. This was determinedby equilibrium
dialysis(Fersht, 1975).
Analysis of Kinetics. The first-orderrate constantsfor the
hydrolysisof E.Tyr-AMP (kJ are comparablein somecases
to those for its formation (k1, eq 3a). The observedrate
kr
e
.;,
*;'
kh
E.Tyr-AMP + PPi --;
E * Tyr + AMP (3a)
constant for the formation of E.Tyr-AMP kobrdis given by
kobsd= kr + kh
(4)
and the steady-stateconcentrationof E.Tyr-AMP ([E.TyrAMPlss) by
(5)
[E.Tyr-AMP]ss = lBlolh/(kr + ftr,)]
where [E]e is the total concentrationof enzyme. [k6 is the
sum of the rate constantsfor dissociationof the adenylateand
direct hydrolysis(Wells et al., 1986).1[E]s may frequently
be measuredby [E.Tyr-AMP]55 at saturatingconcentrations
of substrates.Alternatively,the enzymewas assayedby its
A2ssas we find that mutants are generallygreater than gOVo
activeon activesite titration. Thus
kr = kou.a[E.Tyr-AMP]ss/[E]o
(6)
For reactionswhich are too slow to follow to completion,the
valueof /<lmay be calculatedfrom the initial rate of formation
of E.Tyr-AMP 26,since
(7)
vs = kçlBlo
(Inorganic pyrophosphatase
is added to removeany pyrophosphatethat is producedand would otherwiseobscurethe
kinetics.)
Rssulrs
Choice of Mutations. The most conservativemutation
shouldbe madewhenconductinga mutationalanalysisof the
role of sidechainsin catalysis,removingthe minimal amount
of side chain and minimizing the change in properties.
Truncating lysine residuesto asparagine,and argininesto
glutamines,has the advantageof retainingsomeof the hydrophobicportion of the sidechain and a polar end. This is
idealfor a surfaceresiduethat hasno otherrole than to present
a polar functionto water. However,mutation to asparagine
and glutaminecould give artifactsbecausepolar groupsare
introducedthat can makespuriousinteractionswith substrates
and within the enzyme. We have,accordingly,constructed
a further set of mutantsin which the sidechainshavebeen
cut right back by mutation to alanine. As is seenbelow,there
are significantdifferencesbetweenmutating to Ala and mutating to Asn in two examples.
Mutation Dramatically Alters ReactionRates. Mutation
of eachof the four residuescauseslarge decreases
in the rate
of formation of enzyme-boundtyrosyl adenylatewithout
concomitantdecreases
in its stability: I mol of tyrosyl adenylate accumulatesper mole of enzymedimer with /172of
many secondsto severalminutes. This allows the direct
measurementby pre-steady-state
kineticsof the formation of
enzyme-boundtyrosyl adenylatewith manual samplingand
likewisethe pyrophosphorolysis
rates of the enzyme-bound
complexes(Table I). The availability of pre-steady-state
measurements
allowsconfidentand accuratedetermination
of the activity of debilitatedmutants whoseactivity under
steady-stateconditionscould be obscuredby contaminantsor
revertants.
The effect of mutation on the energylevelsof eachof the
intermediatesis seenreadily from the differenceenergydiagrams(Figure 1), constructedasdescribedby Wellsand Fersht
(1986). We first deducefrom the kineticshow residues82,
86,230, and 233 interactwith the substrates,
transitionstates,
and intermediatesand then correlatethe interactionswith the
crystal structure.
Lys-230 and Lys-233. Mutation to either alanineor asparaginegivesvery similar resultsfor each residue. In all
cases,the energylevelsof the E.Tyr and E.Tyr-AMP complexesare eithervirtually unaffectedor only slightly altered
whereasthe energylevelsof the transition-stateand E.TyrAMP.PPi complexesare considerablyraised. The dissociation
constantof magnesiumpyrophosphatefrom the complexis
raisedabovethe solubilityand so becomesimmeasurablyhigh.
The binding of ATP to the E.Tyr complexesof mutants
Ala-230and Asn-230is only slightly weakened.The direct
interpretationof the differenceenergiesis that Lys-233binds
stronglyto ATP, the transitionstate,and pyrophosphate
while
Lys-230appearsto bind stronglyto the transitionstateand
pyrophosphate
only.
A19-86and Lys-82. Both mutantsof A19-86haverelatively
small changesin the bindingof Tyr, ATP, and Tyr-AMP, but
thereis a large destabilizationof the transitionstate. Interestingly,althoughkulKpp is reducedto a similar low value
in both cases,the valueof Koois increasedfrom 0.61 mM for
wild-typeenzymeto2.5 mM for Gln-86and becomes)10 mM
1584 BrocHEMrsrRY
FERSHT
âo
ET AL.
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ncuru l: Differcnceenergydiagrâms(freeenergyof mutântcomplex- freeeûergyof wild-typeenzymecomplex)for mutationof Lys Ala-82or Asn-82(top right),Arg - Ala-86or Gln-86(top left), Lys - Alâ-230or Asn-230(bottomleft), ândLys * Ala-233or Asn-233
(bottomright).
for Ala-86. The differencebetweenAla-86 and Gln-86 in
is what would be expectedif Gln-86
binding pyrophosphate
is able to bind pyrophosphate
but in a nonproductivemode.
Classicalnonproductivebinding lowersindividual valuesof
ko, and Ky in the Michaelis-Mentenequationwithout altering
their ratio. This illustratesthe artifactsthat can arisewhen
side chainscontainingfunctional polar groupsare used as
replacements
in mutagenesis.Arg-86 thus bindsthe transition
state and pyrophosphate.
Mutation of Lys-82 is lessclear cut. For both mutants,
transition-state
bindingis weakenedby 3 kcal/mol. Further
for Ala-82, but not Asn-82, ATP binding is weakenedsignificantly and pyrophosphatebinding is greatly weakened.
Sinceartifactsfrom spuriousbindingare likely to arisefrom
the amide group of Asn, the resultsfrom Ala-82 probably
reflect the binding propertiesof Lys-82. This appearsto bind
tightly and ATP weakly.
the transitionstateand pyrophosphate
DrscussroN
The results show clearly that residuesLys-82, A19-86,
Lys-230,and Lys-233interactwith the transitionstatefor the
with
formationof tyrosyl adenylateand with pyrophosphate
relativelyminor effectson the binding of tyrosineand tyrosyl
adenylate(Figure 1). Further, Lys-82 interactsweaklyand
Lys-233 interactsstronglywith ATP.
Correlation with Crystal Structure. The involvementof
Lys-230 and Lys-233 in binding and catalysisis entirely
unexpectedfrom examinationof the crystal structure. Residues82, 86, 230, and 233are on two exposedloopswhich have
very high temperaturefactors(Brick & Blow, 1987). The
temperaturefactor B is a measureof the disorderof atoms,
that is, their equilibriumoccupancyof differentconformational
statesand/or their randommotion betweensuchstates.The
terminal guanidiniumnitrogensof A19-86haveB valuesof
nearly 50 and so are on the borderlineof beingvisiblein the
electrondensitymap. The sidechainsof Lys-230and Lys-233
are so mobile that they are not observablein the map. The
long sidechainsof lysineresiduesare frequentlymobile on
the surfaceof proteins,and so it is not uncommonfor the
positionsof the e-NH3+groupsto be indeterminate.However,
a plot of the averagevalueof ^Bfor the peptideC and N, and
the C. of eachresidue(Figure 2), which givesa measureof
the displacementof the backbonefrom its mean position,
revealsthat residuesLys-82and A19-86are in a regionof high
mobility (residues81-89,peakingat A19-86). Lys-230and
Lys-233are in an extendedstretchof mobilebackbonefrorn
residue222 to residue238,with exceptionalmobility (residues
230-237,peakingat Glu-235).
Irrespectiveof the uncertaintyin the positionsof Lys-230
and-233,it is clear that there are sidechainstoo far from the
substratesfor direct interactionwithout considerablymoveand -233
ment of the loop. The a-carbonatomsof lysines-23O
of
tyrosyl
adenylate.
are 16-17 L from the a-phosphate
Building the sidechainsof the lysinesin the most extended
conformation possible,pointing toward the model of the
transition state. doesnot allow direct contact: the closest
distanceof approachof the e-NH3+groupsof eitherLys-230
or Lys-233is about 8 Â from an oxygenion of the 7-phosphate, and they are further away from the a- and B-phosphates. These distancescannot be sensiblyshortenedby
moietyof ATP toward Lys-230and
movingthe pyrophosphate
Lys-233for two reasons.First, we know the positionof the
7-phosphatein the transitionstatewith somecertaintyfrom
the earlier studies. Second,movementtoward Lys-230and
groupsawayfrom Lys-82
Lys-233woulddraw the phosphate
moietycannotbind simuland Arg-86-the pyrophosphate
taneouslyto Lys-82and A19-86on onesideand Lys-230and
Lys-233on the other.
TYROSYL-TRNA
SYNTHETASE
40
MECHANISM
factors(Leatherbarrowet al., 1985). The proximitybetween
the transitionstateand the two residueswas found when the
coordinatesof the transition state were transferredto the
refinedstructure(Brick & Blow, 1987)without any adjustment (R. J. Leatherbarrow,unpublishedresults).1
It is extremelyunlikely that the interactionswith Lys-230
and Lys-233couldarisefrom long-rangeelectrostatic
effects:
such effectswould not have the observedspecificityfor the
transitionstate comparedwith ATP, and the experimental
energiesare far higherthan for electrostaticeffectsrecently
measuredin the activesiteof subtilisin(Russellet al., 1987).
MechanisticInterpretation.The four residuesmust interact
with ATP, the transition state, and pyrophosphateby the
enzymewrappingitself aroundthe pyrophosphate
moiety;that
is, thereis an induced-fitmechanism.The minimal reaction
schemeis that ATP bindsto the enzyme-tyrosine
complexand
draws in Lys-82 and Lys-233 (Figure 3). At that state,
interactionsare not made with A19-86 and Lys-230 (this
study) and alsonot with Cys-35,His-48 Thr-40, His-45,and
Thr-51 (Wells & F'ersht,1986;Ho & Fersht,1986). Strong
interactionsare made,however,betweentheseresiduesand
the transitionstate(Figure3). Stronginteractionsare also
madebetweenthe pyrophosphate
and Thr-40,His-45,Lys-82,
A19-86,Lys-230,and Lys-233in the E.Tyr-AMP.PPicomplex.
In the E.Tyr-AMP complex,Lys-230
and Lys-233moveback
to their positionsin free enzyme,A19-86 presumablyalso
moves,and Lys-82 binds to the a-phosphatevia a water
molecule.
Likelihood of Additional Intermediate betweenGround
State and Transition State. It is expectedfrom chemical
reasoningthat thereis an intermediatebetweenthe E.Tyr.ATP
complexand the transitionstate. It seemsunlikely from the
principle of least motion that the large changein enzyme
structureon forming the transitionstate and the formation
of the chemicalbond betweentyrosineand ATP would occur
simultaneously.It is more likely that there is a high-energy
intermediatebetweenE.Tyr.ATP and E[Tyr-ATP]+ in which
the structureof the enzymeand the interactionsbetweenit
and the substrateresemblethoseof the transitionstate (eq
8). The groundstateor "open" complexdoesnot make the
E.Tyr.ATPlop"n;
e E.Tyr.ATP1"ro,.o;
= E[Tyr-ATP]* (8)
Arg-86
10
40
60
BO
Residue
0
Glu-235
Lys-233
Lys-230
10
0
210
230
250
270
290
voL. 27, NO. 5, 1988 1585
31 0
Residue
FIGURE
2: Crystallographic
temperature
factorsfor mainchain.B
valuesaveraged
for backbone
C, N, andCo takenfrom Brickand
Blow(1987)andtheirunpublished
data.
The positionof Lys-82 is the clearestfrom crystallographic
studies. Lys-82 in the E.Tyr-AMP complexappearsto be
coordinatedto the a-phosphateof Tyr-AMP via a hydrogen-bondedbridging water moleculebetweenthe e-NH3+
group and an oxygenatom (Brick & Blow, 1987). (This
explainswhy the binding of Tyr-AMP is weakenedon mutation to Ala-82.) The e-NH3+groupof Lys-82is about 3 Â
from an oxygenatom of the B-phosphate
in the modelof the
transition state. The guanidiniumgroup of A19-86 can be
placed reasonablycloseto the B- and 7-phosphatesin the
modeledtransition-statecomplex,at about 3 Â from an oxygen
ion of the 7-phosphateand 3-4 Â from an oxygenion of the
B-phosphate.[It is worth noting that the transitionstatefor
the reactionwasmodeledoriginallyinto an earlier,less-refined,
model of the enzymein which the positionsof Lys-82 and
A19-86 were not known becauseof their high temperature
energeticallyfavorablehydrogenbonds/saltbridgesbetween
it and ATP. Theseare formedin the high-energyor "closed"
complex,and their energiespartly offsetthe energythat has
it to be overcometo causethe conformationalchangesand
distortionsin the enzyme(Figure 4). The existenceof the
opencomplexwould alsoexplainwhy Cys-35doesnot make
a bond with ATP in the E.Tyr.ATP complex: it doesnot bind
until the closedcomplexis reached.
Missing from the reactionschemeis the Mg2+ ion: the
substratesfor the reactionare Mg.ATP in the formation of
E.Tyr-AMP and Mg.PPlin its pyrophosphorolysis.
Now that
it has beendemonstratedthat mobile loopsbring previously
unsuspected
sidechainsinto play,we havebeguninvestigating
further residuesin thoseregions. We have now found that
Glu-235is involvedin the bindingof ATP. Mutation of Glu
- Ala-235weakensthe bindingof ATP to the enzymeso that
the dissociation
constantbecomesimmeasurably
high (>10
timesthe valuefor wild-typeenzyme;J. W. Knill-Jonesand
A. R. Fersht,unpublished
data) and raisesthe energyof the
transitionstateby 1.8kcal/mol. Glu-235couldwell be involvedin bindingthe coordinatedmagnesiumion in the E.
Tyr.ATP complexesand in the transitionstate. The involvementof Glu-235in bindingis yet more evidencefor the
catalyticrole of the mobileloop,and the bindingenergybe-
FERSHT
1586 BrocHEMrsrRY
,nr
A19-96 \N,,
.,
\"*:*,
\
l:.
-
Arg'8â..,
I
oH
'î'1
ôy
o
o
\t
HNJ
wr ur-.-Ï.u.
Nl'
Lys-gl
cln-173-ciNH'__
-o---__H\
o t / o ^r\"o
* s
oarf'-
,
---
..-.''
Â
",,1-r,{733
î
'
1.. ^t \
* u
Asp-78-cQ---- ,/
i'
tÂ"i'(Çrhr.51
'r'r16e-oH
U
i
i*"i
ô
O
I
/>ô*
i-:4,/
oa
,fo',,
"
\.
Cvs-35- sH
His-48
Asp-l76-cq--'
\.
Tyr-34- on
,/o
Cys-35-sH
His-45
Thr-40
ïhr-51
His-48
E[Tyr-ATr1*
E.Tyr.ATP
Arg-86.r, r.*"
\ï2
Y ffi-.;t.""
\ Fi "51
o."
'
,\o.t--"';',/.:7
ryrlos-o'
Ho
L.{
t\.
Asp-176-cÇ--TYr-34- oH
",i
Gln-l73-c(o-l---n.
---H-N-CCq-P-O
Asp-78-cd--'?-i:"
-- "(j
"'...
",i_rP3o
!ri.
- -^-'
His45
tn'fo
\rr,r,
.u=*r"'i;...
Arg-86a^
-NH
His-45
#
_à
\-ro"
lys-az---*"""...
-fo'::::
Grn-170-c(NH,
-o-----n.*"
ET AL.
i){"--.r-;;t$t'
.
Lys-f3O
HrN.w
*
Lvs-233
HrNv/.<_,/
Gln-173- c(
,o r\o----
Tyr-34-oir
Cys-35-
"
\,
Cys-35- srr
sH
E.Tyr-AMP
B.Tyr-AMP.PP;
FrcuRE 3: Bindirg events during the activation of tyrosine. (Top left) Ground-state E.Tyr.ATP complex: ATP binds to Lys-82 ^nd Lys-233
and has insignificantbinding encrgywith Cys-35,Thr-40, His-45, His-48, Thr-51, Lys-82, A19-86,and Lys-230. (Top ght) Transition-state
complex: the charged and other groups interact with the pyrophosphatemoiety of ATP and its ribosering. (Bottom left) E.Tyi-AMP.PPi
complex: the groups still interact with the intermediate. (Bottom right) E.Tyr-AMP complex: the Lys-230/233 loop movesaway from the
aderylate after dissociationof PPi, and Lys-82 binds to the a-phosphateof Tyr-AMP via a bridging wâter molecule(Brick & Blow, 1987).
EITyr-ATP]
EITyr-ATP]
E.Tyr-AMP.PPi
FIGURE
4: Free energyschemefor induced-fit process.(Upper curve)
Hypotheticalenergyprofile for formationof transitionstatein the
absenceof binding energywith enzyme. (Lower curve) Energy profile
using binding energy,assumingthe intermediacyof a high-energy
conformationof ATP (E.Tyr.ATft6"6) which resemblesthe structure
of the transitionstatethat is stabilizedby interactionswith the enzyme.
the interactionenergy
The activationenergyis loweredby AG6;n6inn,
of the transitionstatewith the enzyme,corirparedwith that in the
E.Tyr.ATPopen
coûpl€x.
tween Glu-235 and ATP provides additional binding energy
for moving the loop to the substrate.
Why the Induced Fit? An enzyme catalyzes a reaction by
specifically binding the transition state of the reaction. But,
herethe free enzymeis not complementaryin structureto the
transition state but has to be distorted. Distortion of the
structureof the enzymein the transitionstatecostsenergyand
so is deleteriousto catalysis.The enzymeis facedwith two
problems,however: that of surroundingthe transitionstate
chargedgroupsto stabilizeit and alsoof
with the necessary
allowingsubstratesaccessto the activesite. The compromise
is to havean openactivesite with binding groupsin flexible
regionsof the proteinand to usesomeof the binding energy
of the substrateto distort the enzymeon binding.
M agnitude of Tr ansit ion-S t ate St abi Ii zati on. The energies
in the differenceenergydiagramsare equalto -AAGupp,the
changein free energybetweenwild-typeand mutant enzymes
from one stateto the next alongthe reactionpathwaydescribed
paper(Fersht,1988). It appearsat first
in the accompanying
sight from the differenceenergydiagram(Figure 1) that the
four residues
are stabilizingthe transitionstateby some15-18
kcal/mol. This wouldbe in additionto the 8 or 9 kcal/mol
seenin differenceenergydiagramsfor mutationof Thr-40 and
et al., 1985;Leatherbarrow& Fersht,
His-45 (Leatherbarrow
1987). The sum of thesevaluesis too high to representstabilization of the transition state as they would give a rate
couldbe for
or greater.The discrepancy
of 1017
enhancement
two reasons. First, additivity could simply break down for
multiplesubstitutions.Second,althoughAAcuppis generally
Biochemistry 1988, 27.r587-r59r
a reliable measureof changesin binding energybetweenthe
enzymeand substrateand the stabilizationof one staterelative
to a previousone,there is an important exception.This is when
groupsin the ES complexshed their hydrogenbondswith
water to form bonds with each other (Fersht, 1988). As
emphasized
in the accompanying
paper(Fersht,1988),deletion
of a hydrogen-bond
donorto a chargedgroupwill considerably
overestimate
the stabilizationenergy. This could well be the
casehere.
AcrNowr-EDGMENTS
We thank ProfessorDavid Blow and Dr. Peter Brick for
continual accessto the coordinatesof the tyrosyl-tRNA
synthetaseduring its rehnementand Paul Deer and peter Jones
for technicalassistance.
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H., & Winter, G. (1985)NucleicAcids
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Fersht,A.R., Wells,T. N. C., & Leatherbarrow,
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(1986b) Nature (London) 322, 284-286.
Fersht,A. R., Leatherbarrow,
R. J., & Wells,T. N. C. (1987)
Biochemistry26, 6030-6039.
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Leatherbarrow,R. J., Fersht,A. R., & Winter, G. (1985)
Proc.Natl. Acad. Sci. U.S.A.82,7840-7944.
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Winter, G. (1985)Biochemistry24,5106-5109.
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SpecificLabeling of the Essentialcysteine Residueof l-Methionine 7-Lyase with
phosphateT
a Cofactor Analogue,N-(Bromoacetyl)pyridoxamine
Toru Nakayama,NobuyoshiEsaki,Hidehiko Tanaka,and Kenji Sodar
Institutefor ChemicalResecrch,Kyoto UniDersity,uji, Kyoto-Fu 611,Japan
Receiued
June22, 1987;ReuisedManuscriptReceioed
September3, lgg7
ÀBsrRAcr: r-Methionine TJyase lrom Pseudomonas
putida is compsed of fow identical polypeptidechains
and containsfour cysteinylresiduesper subunit. Wè havefound ôneof them catalyticailyessêntialby its
specificcyanylationwith 2-nitro-5-thiocyanobenzoic
acid. We haveshownits esientialitvalso with N(bromoacetyl)pyridoxamine
5'-phosphate(BAPMP), which is a cofactoranalogueandalsoan jfiinity-labeling
agent' The kinetic data showthat th€ apoenzyme
forrnsa binary cornplexwith BAPMP pdor to covalent
binding. The stoichiometryof inactivationwas I mol of BAPMP pei subunit. We havéshownthat the
cysteineresiduemodifiedwith BAPMP is identicalwith that labeledspecificallywith [laC]iodoacetic
acid.
The arninoacid ssquences
of the peptidescontainingthe essentialcyiteineresidueand tÉe lysineresidue
to which pyridoxal 5'-phosphateis boundweredeterminedby automatedEdmandegradatiôn.
t--Methionine7-lyase(EC 4.4.1.11)is a pyridoxal-prenzyme that catalyzesthe a,7-eliminationof l-methionine to
a-ketobutyrate,methanethiol,and NHl. We havepurified
the enzymefrom Pseudomonas
putida and characterizedit
(Esa k iet al. 1979, 19 8 4 ,1 9 8 5
N;a k a y a mae t a l ., 1 9 8 4 ).W e
havestudiedthe mechanismof inactivationof the enzymeby
a suicidesubstrate,L-propargylglycine,
as well and shownthat
r This work was supported in part by special coordination
funds of the
Ministry of Agriculture, Forestry and Fisheriesof Japan.
a nucleophilicamino acid residuelocatedat the active site is
modifiedwith l-propargylglycine(Johnstonet al., 1979). The
presenceof an essentialcysteineresidueof the enzymewas
I Abbreviations: pyridoxal-P, pyridoxal
5,-phosphate;BApM, N(bromoacetyl)pyridoxamine; BAPMP, N-(bromoacetyl)pyridoxamine
5'-phosphate;NTCB, 2-nitro-5-thiocyanobenzoicacid; DTT, dithiothreitol; DTNB, 5,5'-dithiobis(2-nitrobenzoic
acid); EDTA, ethylenediaminetetraacetate;HPLC, high-performance liquid chromatography;
PTH, phenylthiohydantoin;Tris-HCl, tris(hydroxymethyl)aminomethane
hydrochloride.
0006-2960
@ 1988American ChemicalSocietv
/8810427-1s87$01.50/0