Supplementary Material for Chemical Communications
This journal is © The Royal Society of Chemistry 2005
Supplementary data
Inactivation of Human Angiotensin Converting Enzyme by Copper Peptide
Complexes Containing ATCUN Motifs.
Nikhil H. Gokhale and J. A. Cowan*
Evans Laboratory of Chemistry, Ohio State University, 100 West 18th Avenue,
Columbus, Ohio 43210
SUPPORTING MATERIAL
1.
Materials and Methods
1.1 Determination of Kinetic Constants. Determination of Km and Vmax values was
carried out using the same fluorescence assay as described earlier. Reactions were carried
out for 30 min at 37˚C. Initial rates for the hydrolysis of substrate (Vo) were determined
by following the change in fluorescence (relative fluorescence units/min, RFU/min),
plotted as a function of substrate concentration ([S]) and fit to the Michaelis-Menten
equation Vo = Vmax [S]/ ([S]+Km), using Origin software (Microcal) to determine Km.
Substrate concentration was varied from 0 to 20 µM (Figure SM1 to SM4).
1.2 Determination of IC50 values for rhACE inhibitors using, Mca-Arg-Pro-ProGly-Phe-Ser-Ala-Phe-Lys(Dnp)-OH substrate. Reaction mixtures (100 µL) contained
50 mM HEPES buffer, 300 mM NaCl and 10 µM ZnCl2 (pH 7.4), 10 µM substrate and
1nM (13.1 ng) rhACE. Concentrations of GGH, KGHK, YIHPF, Cu2+, Cu(GGH)-,
Cu(KGHK)+, Cu(YIHPF)-, were varied from 0 to 100 µM. Prior to the start of the
enzymatic reaction (by addition of substrate) the inhibitors were pre-incubated with the
enzyme for 45 min. Reactions were run in the wells of polystyrene 96-well microplates
(Porvair) as described previously. Fluorescence was measured for 70 min. and the
background rate determined for samples containing no rhACE was subtracted from all
reactions to calculate the initial rates in RFU/min. Initial rate data were plotted as
percentage activity, relative to uninhibited control reactions, as a function of inhibitor
concentration (Figure SM5 to SM9).
1.3 Determination of the Type of Inhibition. Reaction mixtures (100 µL) contained
50 mM HEPES buffer, 300 mM NaCl, 10 µM ZnCl2 (pH 7.4) and 1nM (13.1 ng) rhACE.
Two sets of reactions were performed at distinct substrate concentration (5 and 10 µM)
with varying concentrations of inhibitor (0 to 20 µM). Prior to the start of the enzymatic
reaction (by addition of substrate) the inhibitors were pre-incubated with the enzyme for
45 min. Reactions were run in the wells of polystyrene 96-well microplates (Porvair).
Fluorescence change was measured for 30 min as described previously. Initial rate data
were plotted versus inhibitor concentration and a Dixon plot (inverse of initial velocity
versus inhibitor concentration) was generated using Origin (Microcal).
1.4 Determination of Inhibition Constants.
For the competitive inhibitors the
enzyme-inhibitor dissociation constant (KI) was determined by graphical methods by use
Supplementary Material for Chemical Communications
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of a Dixon plot, as well as by the equation below, where IC50, [S] and Km have units of
molar concentration.
KI = IC50 / (1+[S]/Km)
1.5 Determination of the Inhibition Mechanism under Oxidative Reaction
Conditions. Enzymatic reactions performed in the presence of dioxygen and ascorbate
are defined as oxidative conditions, while all other reactions described herein that
contained no ascorbate are defined as hydrolytic. Reaction mixtures (100 µL) contained
50 mM HEPES buffer, 300 mM NaCl, 10 µM ZnCl2 (pH 7.4), 10 µM ascorbate (Lascorbic acid, prepared in deionized water) and 1 nM (13.1 ng) rhACE. The
concentration of inhibitor was set at KI. Prior to the start of the enzymatic reaction (either
by addition of substrate or the enzyme) the inhibitors were pre-incubated with the
enzyme for 45 min. Two sets of reactions were run in the wells of polystyrene 96-well
microplates (Porvair) and fluorescence change (relative fluorescence units/min,
RFU/min) was measured for 30 min as described previously.
Set I: Monitoring the progress curve for the enzymatic reaction under oxidative
conditions:
Following prior pre-incubation of the enzyme with the inhibitor, the enzymatic reaction
was initiated by simultaneous addition of the substrate and ascorbate, and the progress of
the reaction monitored by formation of product as reflected by the observed change of
fluorescence. The initial velocity (Vo), obtained from the progress curve in the presence
and absence of the inhibitor, was plotted versus time as described. Respective control
reactions had either ascorbate present or absent (Figure SM10 and Figure SM11).
Set II: Enzyme deactivation under oxidative conditions in the presence and absence of
inhibitor:
To obtain a true rate of enzyme deactivation by the inhibitor under oxidative conditions
the enzyme was pre-incubated with the inhibitor for 45 min. Ascorbate was added,
followed by a preincubation time, and aliquots of the reaction mixture were taken at
various time intervals and the residual enzyme activity estimated using the 96-well plate
assay described previously. Initial velocity (Vo) obtained in the presence and absence of
inhibitor was plotted as a function of time and fitted to the first-order rate equation to
obtain kobs.
Supplementary Material for Chemical Communications
This journal is © The Royal Society of Chemistry 2005
Figures for Supplementary Material.
600
5 µM Substrate
10 µM Substrate
20µM Substrate
30µM Substrate
40µM Substrate
50 µM Substrate
RFU
(background corrected)
500
400
300
200
100
0
0
5
10
15
20
Time (MIn)
25
30
Figure SM1
Initial Velocity (RFU/Min)
20
15
10
5
0
0
10
20
30
40
Flurogenic Peptide substrate (µM)
Figure SM2
50
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800
0µM substrate
2µM substrate
4µM substrate
5µM substrate
8µM substrate
12µM substrate
14µM substrate
16µM substrate
18µM substrate
20µM substrate
700
600
RFU
500
400
300
200
100
0
0
10
20
30
40
50
Time (Min)
Figure SM3
0.20
-1
1/v (RFU .min)
0.16
0.12
0.08
0.04
Km = 6.6 µM
-1/Km = -0.15
-0.2
0.00
-0.1 0.0
0.1
0.2
0.3
-1
1/[s] (µM )
Figure SM4
0.4
0.5
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Dose Dependence GGH
200
140
120
100
80
GGH
0 µM
4 µM
6 µM
8 µM
10 µM
20 µM
120 µM
150
RFU
160
RFU
200
0 µM
2 µM
4 µM
6 µM
8 µM
10 µM
20 µM
40 µM
80 µM
120 µM
180
Dose Dependence KGHK
100
KGHK
60
40
50
20
0
0
10
20
30
40
50
Time (min)
60
70
80
0
0
10
20
30
40
50
Time (min)
60
70
80
Dose Dependence YIHPF
0 µM
4 µM
6 µM
8 µM
10 µM
20 µM
80 µM
100 µM
120 µM
200
RFU
150
100
3.0
YIHPF
2.5
RFU (initial velocity)
250
50
2.0
1.5
1.0
GGH
KGHK
YIHPF
0.5
0
0
10
Figure SM5
20
30
40
50
Time (min)
60
70
80
0.0
1
10
Concentration (µM)
100
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For Cu2+
+2
Dose Dependance (Cu )
+2
0 µM Cu
+2
2 µM Cu
+2
4 µM Cu
+2
6 µM Cu
+2
8 µM Cu
+2
10 µM Cu
+2
20 µM Cu
+2
40 µM Cu
+2
60 µM Cu
+2
80 µM Cu
+2
100 µM Cu
RFU
300
200
100
% Enzyme Activity
400
100
80
60
40
20
0
0
10
20
30
40
50
60
70
0
Time (Min)
1
10
+2
[I] (Cu ) (µM)
100
Figure SM6
For Cu(GGH)+
Dose Dependance Cu(GGH)
500
100
-1
0 µM (GGH-Cu)
-1
2 µM (GGH-Cu)
-1
4 µM (GGH-Cu)
-1
6 µM (GGH-Cu)
-1
8 µM (GGH-Cu)
-1
10 µM (GGH-Cu)
-1
20 µM (GGH-Cu)
-1
40 µM (GGH-Cu)
-1
60 µM (GGH-Cu)
-1
80 µM (GGH-Cu)
-1
100 µM (GGH-Cu)
RFU
300
80
% Enzyme Activity
400
90
200
60
50
40
30
20
100
0
70
10
0
0
10
20
30
40
Time (Min)
50
60
70
1
10
-1
[I] (GGH-Cu) (µM)
100
Figure SM7
For Cu(KGHK)+
+
Dose Dependance Cu(KGHK)
400
300
250
200
80
% Enzyme Activity
350
RFU
100
+1
0 µM (KGHK-Cu)
+1
2 µM (KGHK-Cu)
+1
4 µM (KGHK-Cu)
+1
6 µM (KGHK-Cu)
+1
8 µM (KGHK-Cu)
+1
10 µM (KGHK-Cu)
+1
20 µM (KGHK-Cu)
+1
40 µM (KGHK-Cu)
+1
60 µM (KGHK-Cu)
+1
80 µM (KGHK-Cu)
+1
100 µM (KGHK-Cu)
150
100
60
40
20
50
0
0
0
10
20
30
40
Time (Min)
50
60
70
1
10
+1
[I] (KGHK-Cu) (µM)
100
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Figure SM8
For Cu(YIHPF)+
-
Dose Dependance Cu(YIHPF)
500
90
-1
0 µM (YIHPF-Cu)
-1
2 µM (YIHPF-Cu)
-1
4 µM (YIHPF-Cu)
-1
6 µM (YIHPF-Cu)
-1
8 µM (YIHPF-Cu)
-1
10 µM (YIHPF-Cu)
-1
20 µM (YIHPF-Cu)
-1
40 µM (YIHPF-Cu)
-1
60 µM (YIHPF-Cu)
-1
80 µM (YIHPF-Cu)
-1
100 µM (YIHPF-Cu)
300
200
80
% Enzyme Activity
400
RFU
100
70
60
50
40
30
20
10
0
100
0
0
10
20
30
40
50
Time (Min)
Figure SM9
60
70
1
10
-1
[I] (YIHPF-Cu) (µM)
100
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Relative Fluorescence Intensity (RFU)
600
E (Hydro)
E (Oxd)
E + Ki[I] (Hydro)
E + Ki[I] (Oxd)
E + 20xKi[I]
500
400
300
200
100
0
0
20
40
60
Time (min)
Figure SM10. Plot of RFU with time (min) under various experimental conditions (progress
curve), where E(hydro) is the hydrolytic control, E(Oxd) is the oxidative control, E +
K(I)(Hydro) contains the inhibitor at a concentration of 4µM under hydrolytic conditions, E +
K(I)(Oxd) contains the inhibitor at a concentration of KI under oxidative conditions, and E +
20xK(I) ) contains the inhibitor at a concentration 20-fold higher than KI under oxidative
conditions
300
E (Oxd)
E + Ki[I](Oxd)
250
RFU
200
150
100
50
0
0
5
10
15
20
Time (minutes)
25
30
Figure SM11. Plot of RFU with time (min) under oxidative experimental conditions
(progress curve), where E (Oxd) is the oxidative control, E + K(I)(Oxd) contains the
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inhibitor at a concentration of KI under oxidative conditions.