Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
pubs.acs.org/biochemistry
S Supporting Information
*
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Article
Biochemistry
BaIMPDHL. The mechanistic insights should prove to be
useful in the rational design of IMPDH-targeted therapy.
v = v0
[E]0 [I]0 K i* +
(1)
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Article
Biochemistry
The asymmetric condence intervals for adjustable regression parameters (kinetic constants, initial concentrations, and
oset on the signal axis) were determined by using the prof ile-t
search method of Bates and Watts.2931 In the case of steadystate initial rates, the condence level for marginal condence
intervals of model parameters (as opposed to joint condence
regions, which were not evaluated) was 95%. However, in the
case of stopped-ow pre-steady-state kinetic data, the individual
times points are not statistically independent. Therefore, the
critical value of the residual sum of squares was chosen by using
the empirical approach advocated by Johnson.3234 According
to this method, the parameter space is searched until the best-t
residual sum of squares increases by a reasonably large
percentage of its best-t value. All analyses reported here
used a SSQ of 5%.
All data analyses were performed by using the software
package DynaFit.24,35 The DynaFit input script les (model
specication, initial values of nonlinear regression parameters,
and the method of analysis) are listed in full in the Supporting
Information.
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Article
Biochemistry
Scheme 1. Redundant Kinetic Mechanism of IMPDH
Figure 2 shows the evolution of enzyme species concentrations corresponding to the 1 mM NAD+ kinetic trace
displayed in Figure 1. Note again the highly complex shapes of
the plots, essentially defying any possible use of the
conventional multiexponential analysis, as the overall rate of
product formation is proportional to the instantaneous
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Article
Biochemistry
Table 1. Averages (n = 16) and Corresponding Standard Deviations of Microscopic Rate Constants from Replicates Determined
by the Global Fit of Stopped-Flow Transient Kinetic Dataa
unit
k2
k2
k3
k3
k4
k4
k5
k7
k7
k8
k8
k9
k9
M
s1
s1
s1
s1
M1
s1
M1
s1
M1
s1
M1
s1
s1
s1
s1
s1
best t
lower limit
upper limit
0.034 0.001
13 3
91 3
37 10
360000 400000c
3500 4000c
14.4 0.4
0.008 0.002
44 7
12 0.9
12 0.5
4.1 0.5
0.264 0.004
0.03 0.001
4.9 2
81 3
18 6
180 40
1.7 0.4
13.6 0.5
0.0044 0.0006
24 2
8 0.9
9.7 0.4
2.6 0.4
0.213 0.004
0.04 0.002
32 7
110 8
86 40
b
b
15 0.4
b
b
17 1
16 0.6
6 0.6
0.321 0.006
a
The lower and upper limits are asymmetric condence intervals determined according to the method proposed by Johnson3234 at the 5% SSQ
level. See also Figures S4S6. bThe upper limit is undened at the 5% SSQ condence level.33 However, the dissociation equilibrium constants k4/
k4 and k7/k7 were invariant during the condence interval search (see Figure S5). cCoecient of variation (%CV) of >100%. However, the
dissociation equilibrium constant k4/k4 is very well-dened by the data across all 16 combinatorial replicates (see Figure S6).
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Article
Biochemistry
Figure 3. Comparison between observed initial rates (symbols) and initial rates predicted from the theoretical model derived for the stopped-ow
transient kinetic data (curves). For details, see the text.
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Article
Biochemistry
Table 2. Comparison of Predicted and Observed SteadyState Kinetic Constants from a Global Fit of Combined
Initial Rate Data to eq 2, Where Kinetic Constants Are
Dened by eqs S25S33a
parameter
1
2
3
4
5
6
7
kcat, s1
Km(B),
M
Ki(I), M
Ki(I,B),
M
Ki(B), M
Ki(Q), M
Ki(Q,B),
M
observed
standard error
CV
(%)
low
high
11.6
415
11.6 0.2
460 20
1.8
4.0
11.2
430
12.0
490
0.791
0.0572
0.5 0.2
0.051 0.002
43.6
4.3
0.3
0.047
1.5
0.054
6610
301
87.2
7400 400
620 360
97 9
5.3
57.7
8.9
6800
320
84
8100
4600
113
predicted
The values in columns labeled low and high are lower and upper
limits of the asymmetric condence interval,29,31 respectively, at the
90% likelihood level. For further details, see the text.
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Article
Biochemistry
Figure 4. A110 plus XMP double-inhibitor experiment. (a) Determination of the apparent inhibition constant for A110 at various xed
concentrations of XMP. (b) Linear least-squares t of experimentally observed values of K*i vs [P].
CONCLUSIONS
IMPDH controls the guanine nucleotide pool, and thus
proliferation, in virtually every organism. Human IMPDH
inhibitors are used as immunosuppressive, antiviral, and
anticancer therapy, and microbial IMPDHs have emerged as
potential drug targets. Prokaryotic and eukaryotic IMPDHs
bind NAD+ in distinctive sites that recognize very dierent
cofactor conformations.15 This dierence has been exploited to
develop selective inhibitors of CpIMPDH in six dierent
frameworks. The cofactor binding site of CpIMPDH is very
H
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Biochemistry
ABBREVIATIONS
CBS, cystathionine -synthetase; CV, coecient of variation
(%); DE, dierential evolution; IMP, inosine 5-monophosphate; IMPDH, IMP dehydrogenase; BaIMPDH,
IMPDH from B. anthracis; BaIMPDHL, BaIMPDH
Glu92Arg220 deletion mutant; CpIMPDH, IMPDH from
C. parvum; MPA, mycophenolic acid; XMP, xanthosine 5monophosphate.
ASSOCIATED CONTENT
S Supporting Information
*
REFERENCES
Scheme 3
Article
AUTHOR INFORMATION
Corresponding Author
ACKNOWLEDGMENTS
The authors thank Cynthia Tung for assistance with the
synthesis of A110.
I
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Article
Biochemistry
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
Yang Weia,1 , Petr Kuzmicb , Runhan Yua , Gyan Modia , Lizbeth Hedstrom,a
a Departments
Abstract
This Supplementary Information document contains the detailed description of (a) the raw experimental data; (b) the mathematical models; (c) the nonlinear regression procedures. This
information relates to the analysis of both the stopped-flow transient kinetic data and the steadystate initial rate data on the inhibition of BaIMPDHL by the small-molecule inhibitor A110
specifically under experimental conditions where IMP as substrate is present at saturating concentrations. This document also contains input files for the software package DynaFit (Kuzmic,
1996, 2009) which was used to perform all kinetic analyses.
Key words: enzyme kinetics; inhibition; IMP dehydrogenase; B. anthracis; mathematics;
statistics
Contents
1 Introduction
2 Stopped-flow transient kinetics
2.1 Experimental data . . . . . . . . . . . . . . . . . . . .
2.2 Theoretical model . . . . . . . . . . . . . . . . . . . .
2.3 Stopped-flow kinetic results . . . . . . . . . . . . . . .
2.3.1 Representative global data set . . . . . . . . .
2.3.2 Combinatorial replicates . . . . . . . . . . . .
2.3.3 Partially constrained model: Rapid equilibrium
2.4 Pre-steady state kinetics: Summary . . . . . . . . . . .
3
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
3
. 3
. 4
. 8
. 8
. 11
. 13
. 15
author
address: Sanford-Burnham Institute, La Jolla, California
Supporting Information
3.3
3.4
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
17
21
22
22
23
24
25
29
39
4 Responses to Reviewers
39
4.1 Linear vs. logarithmic plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
References
41
Appendix
44
A DynaFit scripts
A.1 Global fit of stopped-flow data . . . . . . . . . . . . . . . . . . . .
A.2 Global fit of initial rates . . . . . . . . . . . . . . . . . . . . . . . .
A.2.1 Predict kinetic constants: Classical rate equation . . . . .
A.2.2 Predict kinetic constants: Tight binding rate equation . . .
A.2.3 Kinetic isotope effect . . . . . . . . . . . . . . . . . . . . .
A.2.4 Apparent Michaelis constant of IMP . . . . . . . . . . . . .
A.2.5 Apparent inhibition constant of XMP . . . . . . . . . . . .
A.2.6 Apparent inhibition constant A110 in dependence on [XMP]
A.2.7 Simulate dependence of Ki for A110 on [XMP] . . . . . . .
A.2.8 Linear fit of simulated Ki vs. [XMP] values . . . . . . . . .
A.2.9 Linear fit of experimental Ki vs. [XMP] values . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
44
44
46
46
47
49
50
51
52
52
53
54
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
55
55
56
56
57
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1. Introduction
The main purpose of this Supporting Information document is to describe in sufficient detail
the statistical and mathematical methods that were used for the analysis of (a) the stopped-flow
transient kinetic data and (b) the initial rate kinetic data related to the inhibition of BaIMPDHL
2
by the small-molecule inhibitor A110. The experimental setup includes simultaneous variations
in the concentration three component species:
1. the cofactor, NAD+ ;
2. the coproduct, NADH; and
3. the inhibitor A110.
All raw experimental data are attached to this report as a separate ZIP archive file, along with
the relevant input files for the software package DynaFit [1, 2] that was exclusively used for data
analysis.
2. Stopped-flow transient kinetics
2.1. Experimental data
We performed eight replicated series of experiments ([NAD+ ] = 0.25, 0.5, ..., 8 mM) in
four separate daily sessions. The experimental series where [NAD+ ] was varied alone, in the
absence of NADH or A110, was replicated four times. In the discussion that follows as well,
as in the attached raw data files, these four series of experiments are designated as replicates
N1 N4 . The series where [NAD+ ] was varied in the presence of NADH was replicated
twice (replicates H1 and H2). The series where [NAD+ ] was varied in the presence of A110
was also replicated twice (replicates I1 and I2). To assure proper randomization and thus
increase the reliability of the regression analysis, groups of two replicated series were purposely
obtained on different days, always starting from fresh stock solutions. The organization of the
stopped-flow experiments is summarized in Table S1. The series replicate labels (N1, I1, etc.)
correspond to CSV raw data file names (N1.CSV, I1.CSV, etc.) that are made available as part
of this manuscript.
session
1
2
3
4
replicate
N1, I1
N2, I2
N3, H1
N4, H2
note
Each dataset for global analysis [3] consisted of 21 kinetic traces, namely, seven traces where
[NAD] was varied in the absence of any additional component; seven traces where [NAD+ ]
was varied in the presence of added NADH (product inhibition); and finally seven traces where
[NAD+ ] was varied in the presence of the inhibitor A110. Given the number of available replicates, there exist 4 2 2 = 16 ways in which a group of 7 traces could be systematically drawn
from the combined data set, namely: 4 ways to select a group of curves with [NAD+ ] only; 2
ways to select a group of curves with added NADH; and 2 independent ways to select a group of
curves with added A110. In this work all 16 combinations of 21 traces were analyzed as statistically independent combinatorial replicates. The combinatorial replication scheme is summarized
in Table S2.
combinatorial replicate
N replicate
H replicate
I replicate
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1
1
1
1
2
2
2
2
3
3
3
3
4
4
4
4
1
1
2
2
1
1
2
2
1
1
2
2
1
1
2
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
hours on a 2.4 GHz microprocessor (Intel-i7). The results are shown in Figure S1. Note that
under conditions where the IMP substrate (S) is fully saturating ([IMP] = 1000 M, >> Km(IMP) )
the free, uncomplexed enzyme species E effectively does not appear.
A
B
P
Q
I
E.B
IMP
NAD+
XMP
NADH
inhibitor
E~P.B
k7 k-7
+B
k3
E
k4
E.A.B
k2
E~P
E-P.Q
+Q k-4
k-3
+I
+B
k-2
E.A
k-9
k5
E~P.I
+I
k-8
k8
+A
saturating
k9
E.A.I
Figure S1: Minimal kinetic mechanism for the the inhibition BaIMPDH by A110. Dots represent noncovalent complexation; the dash character represents a covalent bond. The tilde character stands for either covalent bonding or noncovalent complexation, with the actual state being
impossible to determine by stopped-flow kinetics alone.
(S1)
The species concentrations [Q] and [EPQ] at time t were computed from their initial concentrations at time zero, by numerically solving an initial-value problem defined by the ODE system
in Eqns (S2)(S12).
3 Note that the molar response coefficient is not identical to the extinction coefficient of NADH, but rather it is 1000
times smaller. It is the number of dimensionless mOD units (103 dimensionless absorbance units) per micromole/liter
of NADH generated in the reaction.
d[EA]
dt
(S2)
d[B]
dt
(S3)
d[EAB]
dt
(S4)
d[EPQ]
dt
(S5)
d[EP]
dt
(S6)
d[Q]
dt
(S7)
d[P]
dt
+k5 [EP]
(S8)
d[EPB]
dt
(S9)
d[I]
dt
(S10)
d[EAI]
dt
(S11)
d[EPI]
dt
(S12)
The model equations listed above were automatically derived by the software package DynaFit [2] by using the coding listed below. The full text of the DynaFit script file is listed in
Appendix A.1. In the code fragment below, the [mechanism] section is used within DynaFit to
derive the ODE system Eqns (S2)(S12). The [responses] section is used internally to derive
the regression Eqn (S1).
[mechanism]
E.A + B <==> E.A.B
E.A.B <==> E.P.Q
E.P.Q <==> E.P + Q
E.P ---> E.A + P
E.P + B <==> E.P.B
E.A + I <==> E.A.I
E.P + I <==> E.P.I
...
[responses]
:
:
:
:
:
:
:
k2
k3
k4
k5
k7
k8
k9
k-2
k-3
k-4
k-7
k-8
k-9
Q = 6.22
E.P.Q = 1 * Q
Organization of model parameters
Each global [3] data set, analyzed as a single unit, consisted of 21 kinetic traces of three
different kinds as was described in section 2.1. Each kinetic trace was filtered down to 25 time
points, such that the global data sets each contained 21 25 = 525 data points. The nonlinear
least-squares regression model for each global set of 525 data points contained 36 adjustable
parameters, some of which were global i.e. applicable to all 21 data traces; some of which
were local to individual traces; and some of which were semi-global i.e. applicable jointly
to a particular group of progress curves. This breakdown of regression parameters into three
categories is explained in Table S3.
parameter no.
parameter
type
113
14
15
1636
k2 k9
initial [E.A]0
initial [I]0
offset A0
global
global
semi-global
local
121, shared
121, shared
1521, shared
121, individually
Table S3: Organization of regression parameters into categories. For details see text.
,
,
,
k-2 = 10 ??
k-3 = 10 ??
k-4 = 10 ??
,
,
,
k-7 = 100 ??
k-8 = 10 ??
k-9 = 0.1 ??
using the profile-t method of Bates & Watts [911]. However, rather than relying on the standard
statistical formulas to determine the critical value of the residual sum of squares, we utilized
the empirical method advocated by Johnson [1214]. According to this empirical approach, the
parameter space is searched until the best-fit residual sum of squares increases by a reasonably
large percentage of its best-fit value. All analyses reported here used SSQ = 5%, as shown in
Appendix A.1:
[settings]
{ConfidenceIntervals}
SquaresIncreasePercent = 5
2.3. Stopped-flow kinetic results
2.3.1. Representative global data set
Figure S2 4 shows the results of global fit of 21 combined kinetic traces, for one of 16 combinatorial replicates enumerated in Table S2 (in this case replicate N1H1I1, see listing in Appendix
A.1). The corresponding best-fit vales of model parameters are shown in Table S4. The upper
left panel shows 7 traces where NAD+ was varied alone; upper right panel shows 7 traces where
NAD+ was varied in the presence of added [NADH] = 60 M; the lower left panel contains 7
traces where NAD+ was varied in the presence of added [A110] = 6 M (nominal).
The lower right panel shows the plot of instantaneous reaction rates against reaction time.
Note the complex shape of the instantaneous rate plot, in particular the lag phase between t = 0
and approximately t = 20 msec. Also note a shoulder feature between t = 0.1 sec and t = 0.5
sec. This complexity in the progress curve shape indicates that it is in principle impossible to
apply the standard exponential or multi-exponential analysis of the reaction time course.
In Table S4, most of the adjustable baseline offsets values were omitted for clarity. The low
and high values were determined by the profile-t method [911] at SSQ = 5% according to
Johnsons method [1214]. The results listed in Table S4 show that 11 adjustable rate constants
were determined with relatively low formal standard error, ranging from 4 to 17 percent (as
coefficient of variation, cv). The only two exceptions are the rate constants k4 and k4 , which
characterize NADH dissociation and rebinding, respectively.
Asymmetric confidence intervals
The asymmetric confidence intervals for microscopic rate constants, as determined by the
profile-t method [9] at the empirical 5% SSQ level [14], can be grouped into four categories,
depending on how well each rate constant can be identified from the available data. See section
Rate constant bounds in Results and Discussion of the main manuscript.
In the first category are rate constants k2 , k3 , k5 , k8 and k9 ). The likelihood profiles [4] for
these rate constants were all closed from both sides not only at the 5% SSQ level, but also at
the 10% and 25% SSQ level (results not shown). The coefficient of variation from replicates is
lower than 5%. A representative example profile is shown in Figure S3 for rate constants k2 and
k2 . The fact that the likelihood profiles in Figure S3 are closed from both sides means that k2
and k2 are very well defined by the available pre-steady state kinetic data.
4 In order to alow multiple graphic panels to be displayed in the same illustration, all Figures in this document (EPS
files generated by DynaFit [2]) were optimized for on-screen viewing, in the PDF format, rather than for printing, on
paper. Please use the View ... Zoom ... 150% in your PDF file viewer (e.g., Adobe Acrobat) in order to examine
fine details.
[NADH] = 60 M, [A110] = 0
[NADH] = 0, [A110] = 0
60
40
1000 A340
residuals
0.5
residuals
0.25
0.5
1
2
4
6
8 mM
20
40
20
1000 A340
60
80
0.25
0.5
1
2
4
6
8 mM
0
-0.5
0.01
0.5
0
-0.5
0.1
0.01
time, s
0.1
time, s
[NADH] = 0, [A110] = 6 M
[NADH] = 0, [A110] = 6 M
400
600
0.25
0.5
1
2
4
6
8 mM
200
d(1000 A340) / dt
20
0
1000 A340
40
0.25
0.5
1
2
4
6
8 mM
0
-1
residuals
0.5
-0.5
-1.5
0.01
0.1
0.01
time, s
0.1
time, s
Figure S2: Representative replicate (n = 16) from the least-squares fit. For details see text.
In the second category are rate constants k2 , k3 , k8 and k9 . These four rate constants are
marginally less well determined by the data. The confidence intervals are closed from both ends
(upper and lower limit) but the coefficient of variation is typically between 10% and 25%.
In the third category are the two rate constants that characterize substrate inhibition by NAD+
(k7 and k7 ). The likelihood profiles [4] for these two rate constants were closed from both sides
at the 5% SSQ level, but open from above at the 10% SSQ level. This can be seen in Figure
S4. Indeed at the upper end of the confidence intervals, the likelihood profile curves barely
intersect the 5% SSQ level and the profile become nearly horizontal at that level. In contrast,
the lower end of the empirical confidence interval is very well defined.
Johnson [1214] advocates the use of 10% SSQ as a stringent albeit empirical rule
of thumb to assess the identifiability of a given rate constant. Thus in a highly conservative
interpretation of the likelihood profiles displayed in Figure S4, the conclusion is that the upper
limit are undefined for both k7 and k7 . In other words, the data are consistent with the statement
9
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
...
36
param
k2
k2
k3
k3
k4
k4
k6
k7
k8
k8
k8
k9
k9
[E.A]0
[I]0
offset / 1
offset / 2
...
offset / 21
unit
1 1
M s
s1
s1
s1
s1
M1 s1
s1
M1 s1
s1
M1 s1
s1
M1 s1
s1
M
M
mOD
mOD
...
mOD
initial
0.01
10
100
10
1000
10
10
0.01
100
10
10
1
0.1
2.5
6
0
1
...
12
final std.err.
0.0348
16
88.3
23.1
63000
700
14.39
0.0078
43.8
12.33
11.75
4.4
0.26
2.429
4.78
-0.118
1.213
...
9.864
0.001
2.8
2.4
3.4
> 106
> 105
0.19
0.0014
7.6
0.79
0.56
0.35
0.01
0.024
0.2
0.065
0.068
...
0.094
cv,%
low
high
2.9
17.5
2.7
14.7
>> 100
>> 100
1.3
17.9
17.4
6.4
4.8
8
3.8
1
4.2
55.1
5.6
...
1
0.0307
5.79
80.2
9.86
170
1.7
13.71
0.0043
23.7
8.596
9.391
2.9
0.212
0.0434
43.1
107
57.2
>> 108
>> 106
15.43
0.17
995
16.9
14.76
6.29
0.314
note
(a)
(a)
(a)
Only the lower limit of this parameter could be determined by the profile-t search method [9]. The upper
limit approaches infinity.
Table S4: Best-fit model parameters determined from data depicted in Figure S2.
that k7 must be greater than 4.3 104 M1 s1 (see Table S4) but there is no way of saying just
high high k7 could actually be. Similarly, under the strict 10% SSQ empirical rule, k7 must
be greater than 24 s1 (see Table S4) but there is no way of saying just high high k7 could
actually be. This is the same as saying that the NAD+ substrate inhibition step can be described
as instantaneous equilibration between E P and E PN in Figure S1.
In the fourth category are the two rate constants that characterize product inhibition by NADH
(k4 and k4 ). The likelihood profiles [4] for these two rate constants were closed from below not
only at the 5% SSQ level but also the 10% and 25% SSQ levels (results not shown). However,
the likelihood profiles showed no minimum at all and were perfectly flat to the right of the best-fit
values. This can be seen in Figure S4.
The interpretation of one-sided but otherwise perfectly flat likelihood profiles [4], such as
those displayed in Figure S4, is straightforward. All one can say is that k4 must be greater than
170 s1 (see Table S4) but there is no way of saying just high high k4 could actually be. Similarly,
k4 must be greater than 1.7 106 M1 s1 (see Table S4) but the upper limit is undefined. This
is the same as saying that the NADH product inhibition step can be described as instantaneous
equilibration between E P and E PNH in Figure S1.
Rate constant correlations
Figure S5 shows a plot of all pairs of rate constant values that lie within their respective
confidence intervals as shown in Figure S4 (for pairs of k4 and k4 ) and Figure S4 (for pairs of k7
and k7 ). These correlation plots show that although the individual values of rate constants are
poorly defined by the pre-steady state kinetic data, their ratios, i.e. the corresponding dissociation
10
1.05
search
bounds
best-fit
1.2
SSQrel
1.4
search
bounds
best-fit
SSQrel
0.03
0.035
0.04
0.045
20
k2
40
60
k-2
Figure S3: Empirical likelihood profile for microscopic rate constants k2 and k2 . For details
see text.
equilibrium constants, are very well defined.
For example, in the left-hand panel of Figure S5 the plausible values of either k4 or k4 span
from the lower-left corner of the graph (i.e., from the well defined lower limit of the confidence
intervals) up to values at least four orders of magnitude higher (see also Figure S4). However, the
plot of all plausible values of log(k4 ) against all plausible values of log(k4 ) is a perfect straight
line with a unit slope. Thus the ratio Kd(EP.NH) k4 /k4 is invariant within the confidence
intervals for either rate constant and it is equal to Kd(EP.NH) = 100 M. Similarly all plausible
values of the ratio Kd(EP.N) k7 /k7 lie between 5.3 and 5.7 mM.
Thus, in summary, in the analysis of one of the 16 combinatorial replicates depicted in Figure
S2 we obtained well defined values of:
1.1
1.04
SSQrel
1.02
1.05
SSQrel
1.06
1.08
search
bounds
best-fit
100
10
100
k4
k-4
1.05
SSQrel
1.15
search
bounds
best-fit
1.1
1.15
1.1
1.05
SSQrel
0.01
0.1
100
k7
1000
k-7
Figure S4: Empirical likelihood profile for microscopic rate constants k4 , k4 (top) and k7 , k7
(bottom). For details see text.
rate constants k4 and k4 and k7 and k7 . The lower right panel groups together the best-fit values
the four microscopic rate constants that characterize the interactions of A110.
The results shown in Figure S6 indicate that the best-fit values of all microscopic rate constants except k4 and k4 are very well reproduced across all 16 combinatorial replicates. The
coefficient of variation ranges from less than 5% for the forward rate constants (k2 , k3 and k5 ) to
approximately 20% for the reverse rate constants (k2 and k3 ). Interestingly the best-fit values
of k7 and k7 are also very well reproduced. The coefficient of variation from replicates (n = 16)
is approximately 15% for both k7 and k7 , despite the fact that the upper limit of the confidence
interval is undefined at the stringent 10% SSQ level.
In contrast, the replicated best-fit values of k4 and k4 span at least two orders of magnitude.
This observation is in agreement with the fact that neither k4 nor k4 could be identified from
individual replicates (see Table S4 and Figure S4. However, as before, the ratio k4 /k4 shown in
green in the lower left panel of Figure S6 is well defined by the data. The average (n = 16) value
of Kd(EP.NH) k4 /k4 is 100 M.
12
1000
k-7, s-1
100
1000
100
search k5
search k-5
10
10000
search k3
search k-3
100
1000
10000
100000
1e+006
0.01
k4, s-1
0.1
Figure S5: Correlated values of rate constants that lie within the respective confidence intervals.
Left: correlation between k4 and k4 . Right: correlation between k7 and k7 . For details see text.
2.3.3. Partially constrained model: Rapid equilibrium
The results thus far indicate that all 16 combinatorial replicates produced only a small variation in the best-fit values of the NAD+ substrate inhibition rate constants k7 and k7 . However,
under stringent identifiability analysis [4] requirements based on Johnsons 10% or 25% SSQ
empirical rule [13], the upper limit is undefined for either rate constant. Faced with this apparent contradiction5 we have taken the more conservative view of the pre-steady state kinetic
results and proceeded to investigate the mechanistic scenario whereby both substrate inhibition
by NAD+ and product inhibition by NADH are under rapid equilibrium. The main question
is whether or not by setting the association rate constants k4 and k7 to arbitrary high values
might influence the best-fit values of the remaining microscopic rate constants.
To answer this question, a series of five separate fitting models was constructed (five variants
for for each of the 16 combinatorial replicates) where the bimolecular association rate constants
k4 and k7 were both set to an identical high values and subsequently held fixed in the regression analysis. To arrange for type constrained regression analysis, the [constants] section in the
script listed in Appendix A.1 was modified by removing the question marks next to k4 and k7 .
For example, to set kon = k4 = k7 = 10 M1 s1 , we used the coding below:
[constants]
k2 = 0.03 ?
k3 = 80 ?
k4 = 1000 ?
k5 = 10 ?
k7 = 10
k8 = 10 ?
k9 = 5 ?
,
,
,
k-2 = 10 ?
k-3 = 40 ?
k-4 = 10
,
,
,
k-7 = 50000 ?
k-8 = 10 ?
k-9 = 0.3 ?
; fixed "k-4"
; fixed "k7"
5 It is possible that the apparent very high reproducibility of the best-fit values of both k and k
7
7 is some type of
artifact given that the upper limits do not exist under stringent identifiability requirements.
13
k7
k-7
k7/k-7
k2
k-2
k3
k-3
k5
-2
-1
10
15
replicate no.
NADH product inhibition
15
A110 inhibition
0.5
k8
k-8
k9
k-9
-0.5
k4
k-4
k4/k-4
10
replicate no.
10
15
replicate no.
10
15
replicate no.
unit
kon = 10 M1 s1
kon = 20 M1 s1
kon = 50 M1 s1
kon = 100 M1 s1
k2
k2
k3
k3
k4
k4
k5
k7
k7
k8
k8
k9
k9
M1 s1
s1
s1
s1
s1
M1 s1
s1
M1 s1
s1
M1 s1
s1
M1 s1
s1
0.0315
10.7
84.5
47.4
1150
0.0317
10.8
83.1
45.5
2300
0.0318
10.9
82.3
44.6
5730
0.0318 0.0014
10.9 2.9
82.1 1.9
44.3 9.3
11500 1600
100
13.6 0.25
100
566000 27000
13.9 0.94
11.0 0.4
5.51 0.72
0.270 0.004
k2 /k2
k3 /k3
k4 /k4
k7 /k7
k8 /k8
k9 /k9
M
M
M
M
0.0014
2.9
2.1
9.7
160
10
13.7 0.26
10
56000 2700
13.9 0.94
10.9 0.4
5.65 0.74
0.271 0.004
338
1.78
115
5600
0.788
0.0479
20
13.7
20
113000
13.9
11.0
5.58
0.270
0.0014
2.9
1.9
9.5
330
0.25
5400
0.94
0.4
0.73
0.004
342
1.82
115
5630
0.792
0.0485
50
13.6
50
283000
13.9
11.0
5.53
0.270
0.0014
2.9
1.9
9.3
810
0.25
14000
0.94
0.4
0.72
0.004
344
1.85
115
5650
0.795
0.0488
344
1.85
115
5660
0.796
0.0489
Table S5: Averages and standard deviations from combinatorial replicates (n = 16) for microscopic rate constants appearing in Figure S1 while holding the bimolecular association rate
constants k4 and k7 at various fixed values.
rate constant ratios (i.e., equilibrium constants) listed at the bottom of Table S5.
In conclusion, the minimal partially constrained kinetic model for the pre-steady state kinetic
data is one where both the product inhibition by NADH and substrate inhibition by NAD+ should
be considered to occur under rapid equilibrium approximation. Any particular value of kon =
k4 = k7 higher than approximately 107 M1 s1 provides an equally good description of the
experimental data. The best-fit values of the remaining microscopic rate rate constants appearing
in Figure S1 are insensitive to the arbitrary choice of kon > 107 M1 s1 . This rapid-equilibrium
constrained kinetic model could be graphically represented as is shown in Figure S7. The dashed
arrows represent equilibrium binding steps, for which only the lower limit of kon can be estimated.
2.4. Pre-steady state kinetics: Summary
1. The inhibitor A110 binds predominantly to an enzymeproduct complex, E P.
2. E P is either the covalent intermediate or the noncovalent EXMP complex.
3. The association rate constant for I + E P E PI is 5.5 106 M1 s1 .
4. The dissociation rate constant for E PI I + E P is 0.27 s1 (t1/2 = 2.5 sec).
5. The corresponding Kd is 49 nM.
6. The inhibitor A110 binds more weakly to the enzymesubstrate complex, EA.
7. The association rate constant for I + EA EAI is 1.4 107 M1 s1 .
8. The dissociation rate constant for EAI I + EA is 11 s1 (t1/2 = 0.063 sec).
9. The corresponding Kd is 0.80 M.
10. B dissociates rapidly from EAB (k2 = 11 s1 ; not a sticky substrate).
15
E.B
E~P.B
Kd(EP.B)
+B
Kd(EP.Q)
k3
E
E.A.B
k2
E~P
E-P.Q
k-3
+Q
+I
+B
k-2
E.A
k-9
k5
E~P.I
+I
k-8
k8
+A
saturating
k9
E.A.I
Figure S7: Minimal partially rapid-equilibrium kinetic mechanism for the the inhibition
BaIMPDH by A110. The steps shown in red are assumed to be under rapid rapid equilibrium.
11. The forward hydride transfer rate constants is k3 = 80 s1 (k3 > k2 ).
This necessitates the steady state approximation in the analysis of initial rates.
12. The reverse hydride transfer rate constants is k3 = 40 s1 (Keq = k3 /k3 2).
The forward hydride transfer is slightly favored over the reverse transfer.
13. Hydrolysis of the covalent intermediate and product release is partially rate limiting (k5 =
14 s1 , k5 < k3 for hydride transfer).
14. NADH (Q) is a product inhibitor, with predicted Kd 100 M (rapid equilibrium).
15. NAD+ (B) is a substrate inhibitor, with predicted Kd 6 mM (rapid equilibrium).
3. Steady-state initial rate kinetics
We utilized steady-state initial rate kinetic experiments mainly to check the validity of our
primary results, which are based on the stopped-flow rapid kinetic experiments (see section 2
above).
3.1. Experimental data
The steady-state initial rate data were organized in the same fashion, and obtained under
the same reaction conditions, as the stopped-flow transient kinetics data discussed above. Four
categories of experiments were perfomed at a saturating concentration of the substrate IMP ([S]0
= 1.0 mM):
1.
2.
3.
4.
The nominal concentration of the enzyme, [E]0 = 23 nM, was identical in all initial rate
experiments. However, the amplitude (Vmax ) of presumably identical experiments repeated after
16
k-2
k-3
k-4
k-7
k-8
k-9
This input text corresponds to the kinetic mechanism shown in Figure S1. Note that under
IMP saturating conditions the free enzyme species is effectively the enzymesubstrate complex
E.A. Also note that under IMP saturation the kinetic mechanism turn to Uni Bi, with effectively
only a single substrate (B). These facts are indicated by the following input lines:
...
enzyme E.A
reaction B ---> P + Q
...
When DynaFit processed the input text listed above (see Appendix A.2.1), it automatically
generated the following algebraic expressions and utilized those to perform the least-squares fit
of experimentally observed initial rate data:
17
v = [E]0
N
N
D
(S13)
= n1 [B]
(S14)
(S15)
k2 k3 k4
k2 k3 + k2 k4 + k3 k4
n1
d1
(S17)
d2
k8
k8
(S18)
d3
k2 k3 k4
k5 (k2 k3 + k2 k4 + k3 k4 )
(S19)
d4
k2 (k3 k5 + k4 k5 + k3 k5 + k3 k4 )
k5 (k2 k3 + k2 k4 + k3 k4 )
(S20)
d5
k2 k3 k4 k8
k5 k8 (k2 k3 + k2 k4 + k3 k4 )
(S21)
d6
k2 k3 k4 k9
k5 k9 (k2 k3 + k2 k4 + k3 k4 )
(S22)
d7
k2 k4 (k3 + k3 )
k5 (k2 k3 + k2 k4 + k3 k4 )
(S23)
d8
k2 k3 k4 k7
k5 k7 (k2 k3 + k2 k4 + k3 k4 )
(S24)
kcat =
n1
d4
k3 k4 k5
k3 k5 + k4 k5 + k3 k5 + k3 k4
(S25)
Km(B) =
d1
d4
k5 (k2 k3 + k2 k4 + k3 k4 )
k2 (k3 k5 + k4 k5 + k3 k5 + k3 k4 )
(S26)
n1
kcat
=
Km(B) d1
k2 k3 k4
k2 k3 + k2 k4 + k3 k4
(S27)
18
(S16)
Ki(I) =
d1
d2
k8
k8
(S28)
Ki(I,B) =
d4
d6
k9 (k3 k5 + k4 k5 + k3 k5 + k3 k4 )
k3 k4 k9
(S29)
Ki(B,Q) =
d3
d7
k2 k3
k2 (k3 + k3 )
(S30)
Ki(B) =
d4
d8
k7 (k3 k5 + k4 k5 + k3 k5 + k3 k4 )
k3 k4 k7
(S31)
Ki(Q) =
d1
d3
k5 (k2 k3 + k2 k4 + k3 k4 )
k2 k3 k4
(S32)
Ki(Q,B) =
d4
d7
k3 k5 + k4 k5 + k3 k5 + k3 k4
k4 (k3 + k3 )
(S33)
It is noteworthy that only one of the two A110 inhibition constants (the competitive constant Ki(I) ) is by definition equivalent to the equilibrium dissociation constant of the corresponding enzymeinhibitor complex:
Kd(EA.I)
k8
k8
Ki(I)
k8
k8
In contrast the uncompetitive inhibition constant Ki(I,B) is by definition larger than the corresponding equilibrium constant, as shown in Eqn (S34). See Figure S8 for graphical illustration
of which microscopic rate constant enter into the definition of Ki(I,B) .
Kd(EP.I)
k9
k9
Ki(I,B)
k9
k3 + k3 + k4
1 + k5
k9
k3 k4
!
(S34)
This result is contrary to assumptions that occasionally appear in the biochemical literature.
For example, Biswanger [p. 3][15] writes in a well-regarded enzyme kinetics textbook:
From equilibrium treatments thermodynamic constants, like association or dissociation constants, are derived, while kinetic studies yield the more complex kinetic
constants. On the other hand there are also similarities. [...] [F]or example inhibition
constants, although determined kinetically, are really dissociation constants.
19
E.A.I
E.A.I
=
Kd
E.A
Ki(I)
Ki(I,B)
E-P.Q
E.A.B
E.A
E-P
E.A.B
E-P.B
E-P.Q
E-P
E-P.B
Kd
E-P.I
E-P.I
Figure S8: The red arrows represent all microscopic rate constant that define composite inhibition constant for the kinetic mechanism displayed in Figure S1. The competitive Ki is equal to
the corresponding Kd , whereas the uncompetitive Ki is not.
The IMPDH inhibition mechanism shown in Figure S1 provides a counter-example illustrating clearly that not all inhibition constants are really dissociation constants, although some
are.
The computer-generated initial rate model shown in Eqns (S13)(S24), formulated in terms
of the microscopic rate constants, was converted by standard algebraic manipulations [16, pp.
523-530] into the more conventional form in terms of the composite kinetic constants. The resulting steady-state initial rate law is shown in Eqn (S35), where n and d represent the numerator
and denominator defined by Eqn (S36) and Eqn (S37), respectively.
v =
[E]0 kcat
[B]
Km(B)
1+
n
d
(S35)
(S36)
[I]
[Q]
[B]
[Q][I]
+
+
+
Ki(I) Ki(Q) Km(B) Ki(Q) Ki(I)
[B][I]
[B][Q]
[B]2
+
+
Km(B) Ki(I,B) Ki(B,Q) Ki(Q) Km(B) Ki(B)
(S37)
The kinetic constants kcat , Km(N) , etc., are defined in terms of the microscopic rate constants
appearing in Figure S1 as shown in Eqns (S25)(S33). However, note that there is an inherent
ambiguity in the definition of inhibition constants, as explained by Segel [p. 524][16]:
[Inhibition] constants associated with a given ligand will equal the ratio of two
denominator coefficients. The ratio must be chosen so that, after canceling subscript
letters, the letter corresponding to the ligand associated with the constant remains as
a subscript in the denominator of the ratio. [...] For some systems, it will be possible
to find quite a few ratios of coefficients that satisfy the cancelation requirements
above.
20
Indeed, in the case of the kinetic mechanism depicted in Figure S1 we have to choose between two mutually exclusive formulations of the initial rate equation. One variant is shown
immediately above. Another variant is defined by Eqn (S38), where d0 is an alternate definition
of the denominator.
d0
1+
+
[B]
[B][Q]
[B]2
[Q]
+
+
+
Ki(Q) Km(B) Km(B) Ki(Q,B) Km(B) Ki(B)
[I]
[Q][I]
[B][I]
+
+
Ki(I) Ki(Q) Ki(I) Km(B) Ki(I,B)
!
[B]
[B]
[Q]
[Q]
1+
+
1+
+
Ki(Q) Km(B)
Ki(Q,B) Ki(B)
"
!
#
1
[Q]
1
[B]
+[I]
1+
+
Ki(I)
Ki(Q)
Ki(I,B) Km(B)
(S38)
The difference between the two formulations of the IMPDH rate equation appears in the
particular denominator term that contains the concentration product [B][Q]. In the case of denominator d defined by Eqn (S37), NAD+ (B) is formally treated as a mixed type inhibitor
because it is associated with two inhibition constants, Ki(B) and Ki(B,Q) , whereas NADH (Q) is
treated as an uncompetitive inhibitor because it is associated with only a single inhibition constant Ki(Q) . In contrast, in the case of denominator d0 defined by Eqn (S38), NAD+ is treated
as an uncompetitive inhibitor because it is associated with a single inhibition constants, Ki(B) ,
whereas NADH is treated as a mixed-type inhibitor, because it is associated with two inhibition
constant Ki(Q) and Ki(Q,B) .
These two views of the same kinetic process are both equally correct, but they are mutually
exclusive. Therefore it is meaningless to ask whether NAD+ substrate inhibition in IMPDH
kinetics should be classified as uncompetitive or mixed-type, or equivalently whether NADH
product inhibition is uncompetitive or mixed-type. The only appropriate answer is, it depends
which of the denominator formulations (d or d0 ) is chosen and, importantly, that choice is entirely
arbitrary.
3.2.2. Tight binding rate equation (Morrison)
All steady-state initial rate experiments were conducted at nominal enzyme concentrations
23 nM, based on Bradford assay. The equilibrium dissociation constant k9 /k9 predicted from
the pre-steady state kinetic results is approximately 50 nM (Table S5). When the active enzyme
concentration and the inhibition constants are comparable in magnitude, tight binding (i.e. inhibitor depletion) must be properly taken into account in the mathematical model for the analysis
of initial rates.
Morrison [17] derived a generic form of the enzymatic initial rate law that applies to all enzyme mechanisms where the inhibitor forms any number of 1:1 catalytically inactive complexes
with various enzyme forms. Morrisons original formula6 is reproduced without changes in Eqn
(S39), where [E]0 is the total or analytic concentration of enzyme active sites; [I]0 is the total
6
21
or analytic concentration of the inhibitor; D is the denominator of the initial rate equation derived by the King-Altman method [18] in the absence of the inhibitor; N is the numerator of
the initial rate equation derived by the King-Altman method in the absence of the inhibitor and
assuming that that there is no product formation from any enzymesubstrateinhibitor complex
(i.e., disallowing partial inhibition); Ni is the numerator of the distribution equation for the given
enzymeinhibitor complex [19]; and Ki is the inhibition constant corresponding to the enzyme
inhibitor complex specified by the term Ni .
v =
v
u
u
u
u
u
u
u
u
u
N u
[I]0 [E]0
4 [E]0
1
[I]
[E]
t 1
0
0
+
!+
!
!+
(S39)
2 P Ni
D
D
P Ni
P Ni
Ki
Ki
Ki
When the generic terms appearing in Eqn (S39) (i.e., N, D, Ni , and Ki ) are appropriately
specialized on the basis of the assumed kinetic mechanism shown in Figure S1, we obtain Eqn
(S40), where v0 is the uninhibited reaction rate (observed at zero inhibitor concentration) and Ki
is the apparent inhibition constant [20, 21]. The composite kinetic constants that appear in Eqns
(S41)(S42) are defined in terms of the microscopic rate constants appearing in Figure S1 as is
shown in Eqns (S25)(S33).
v = v0
v0
Ki
[E]0 [I]0
= [E]0 kcat
Ki
q
2
+
[E]0 [I]0 Ki + 4 [E]0 Ki
2 [E]0
[B]
Km(B)
[Q]
[B]
[Q]
[B]
1+
+
1+
+
Ki(Q) Km(B)
Ki(Q,B) Ki(B)
[Q]
[B]
[Q]
[B]
1+
+
1+
+
Ki(Q) Km(B)
Ki(Q,B) Ki(B)
!
[Q]
1
[B]
1
1+
+
Ki(I)
Ki(Q)
Ki(I,B) Km(B)
(S40)
(S41)
!
(S42)
This script performs a highly constrained least-squares fit, such that only the enzyme concentrations are optimized whereas all composite kinetic constants are held fixed at values predicted
from the results of stopped-flow experiments. In particular, the best-fit values of microscopic rate
constants listed in the rightmost column of Table S5 were used to compute the predicted values
of steady state kinetic constants by using Eqns (S25)(S33). These predicted values are listed
in Table 2, main manuscript, column labeled predicted. The regression model proper is the
Morrison Eqn (S40).
Since all kinetic constants are fixed in the highly constrained regression analysis, the fit
largely a comparison between initial rates predicted from the stopped-flow experiment and initial rates observed experimentally. The results are shown graphically in Figure 3 of the main
manuscript. The best-fit values or all adjustable enzyme concentrations are listed in Table S6.
#
par/set
1
2
3
4
5
[E](1)
0 , M
[E](2)
0 , M
[E](3)
0 , M
[E](4)
0 , M
[E](5)
0 , M
inital
0.01
0.01
0.01
0.01
0.01
final std.err.
0.00978
0.00838
0.00921
0.01067
0.00910
0.00007
0.00012
0.00013
0.00010
0.00010
cv,%
Note
0.8
1.5
1.5
1.0
1.1
Table S6: Global fit of combined initial rate data to the system of Eqns (S40)(S42). All steadystate kinetic constants were held fixed at values predicted by the analysis of stopped-flow data.
For further details see text.
The results displayed graphically in Figure 3 (main manuscript) show that the microscopic
rate constants determined by the stopped-flow measurements predict the initial rate data reasonably well. For example the position of the maximum on the NAD+ saturation curve (approximately at [NAD+ ] = 1.5 mM), in the upper left panel, is very well predicted, as is the slope of
the downward portion due to substrate inhibition. In the upper right panel of Figure 3 (main
manuscript), the relative spacing of the NAD+ saturation curves at different concentration of
NADH as product inhibitor is also very well predicted. The lower left panel shows the inhibition
effect of A110 at relatively low concentrations of NAD+ . Again the relative spacing of the substrate saturation curves is very well predicted. The same applies to the right-hand panel of Figure
3 (main manuscript) displaying the inhibitory effect of A110 at relatively high concentrations of
NAD+ .
3.3.2. Predicted vs. observed kinetic constants
In the next round of kinetic analysis, the experimental data displayed in Figure 3 (main
manuscript) were subjected to a least-squares fit in which the kinetic constants defined in terms
of the microscopic rate constants by Eqns (S25)(S33) were treated as optimized parameters.
The required DynaFit notation is exactly identical to the listing in Appendix A.2.2, except for
the fact that the initial estimates of model parameters are marked with question marks:
;
; Enzyme concentrations in different experiments: First one fixed.
;
cE1 = 0.009782 ; based on compare-v script
cE2 = 0.01 ?
23
cE3 = 0.01 ?
cE4 = 0.01 ?
cE5 = 0.01 ?
;
; Extinction coefficient of NADH:
;
rQ = 6.22
; fixed !
;
; Predicted kinetic constants: Get confidence intervals.
;
kcat = 11.6 ??
KmB = 415 ??
KiI = 0.791 ??
KiIB = 0.0572 ??
KiB = 6610 ??
KiQ = 301 ??
KiQB = 87.2 ??
A single question mark identifies a model parameter that should be optimized in the regression analysis. A double question mark identifies a parameter, for which we additionally desire
the asymmetric confidence interval to be computed by using the profile-t method of Bates &
Watts [911]
Note that one of the enzyme concentrations ([E](1)
0 = 9.78 nM) was treated as a fixed parameter set to the best fit value determined in the previous round of analysis, Table S6. This was
necessary to anchor the kcat value because the product Vmax = kcat [E]0 appears in the initial
rate equation and therefore it is impossible to simultaneously determine both kcat and [E]0 from
any particular set of initial rate measurements. The results of fit are shown graphically in Figure
S9. The list of best-fit adjustable model parameters is shown in Table 2, main manuscript. The
column labeled predicted lists the expected values of kinetic constants, i.e., the values computed beforehand using Eqns (S40)(S42) and the numerical values of stopped-flow microscopic
rate constants listed in the right-most column of Table S5. The two sets of kinetic constant values
(predicted from transient kinetics vs. observed from initial rates) are in good agreement within
the experimental error expressed as the 90% confidence-level intervals.
3.3.3. Diagnostic Eadie-Hofstee plots
The Eadie-Hofstee plots corresponding to Figure S9 are shown in Figure S10. To arrange for
automatic generation of Eadie-Hofstee plots by the DynaFit software, the input script listed in
Appendix A.2.2 was modified simply by inserting a single line into the [data] section, as follows:
[data]
transform EH
As a reminder, in the absence of substrate inhibition (i.e., distinct maximum on the substrate
saturation curves) the Eadie-Hofstee plots for enzyme inhibition have the following properties:
1. Competitive: Straight lines intersect exactly on the vertical axis.
2. Uncompetitive: Straight lines intersect exactly on the horizontal axis.
3. Mixed, predominantly competitive: Straight lines intersect in the -X, +Y" quadrant of the
v/[S], v coordinate system.
24
NAD
NAD + NADH
0.4
Q = 0 mM
Q = 0.05
Q = 0.10
Q = 0.15
0.2
v, mOD/sec
0.1
0.2
v, mOD/sec
0.3
0.4
A = 0.75 mM
A = 1 (a)
A = 1 (b)
A = 1 (c)
2000
4000
6000
[NAD+], M
1000
0.3
0.4
I = 0 nM
I = 15
I = 30
I = 60
I = 90
I = 150
0.1
0.2
v, mOD/sec
0.4
I = 0 nM
I = 15
I = 30
I = 60
I = 120
I = 180
0.2
v, mOD/sec
500
[NAD+], M
500
[NAD+], M
1000
2000
4000
[NAD+], M
6000
Figure S9: Global fit of combined initial rate data to the system of Eqns (S40)(S42). One of
the enzyme enzyme concentrations ([E](1)
0 ) was held as a fixed parameter. For further details see
text.
4. Mixed, predominantly uncompetitive: Straight lines intersect in the +X, -Y" quadrant of
the v/[S], v coordinate system.
5. (Pure) Non-competitive: Straight lines are strictly parallel, no intersection point.
In our particular situation the Eadie-Hofstee plots are not linear, because of substrate inhibition by NAD+ . Never-the-less it could be shown that the properties No. 2 (uncompetitive
inhibition) and No. 4 (predominantly uncompetitive inhibition) do apply without change. That
is, even in the presence of substrate inhibition the Eadie-Hofstee model curves would necessarily
intersect on the horizontal axis if the inhibition pattern were uncompetitive.
In actuality there does not seem to be an intersection point on the horizontal axis (v/[S]).
Note in particular the lower left panel of Figure S10. The Eadie-Hofstee model curves do not
intersect on the horizontal axis, which corresponds to mixed-type inhibition by A110.
3.3.4. Predicted vs. observed H/D isotope effect
If NAD+ were a sticky substrate, that is, bound effectively irreversibly to the EIMP complex as previously reported for C. parvum IMPDH [5], the rate constant k2 in Figure S1 would
25
0.4
Q = 0 mM
Q = 0.05
Q = 0.10
Q = 0.15
0.2
v, mOD/sec
0.1
0.2
v, mOD/sec
0.3
0.4
A = 0.75 mM
A = 1 (a)
A = 1 (b)
A = 1 (c)
0.0005
0.001
0.0015
v / [NAD+]
0.001
0.3
0.4
I = 0 nM
I = 15
I = 30
I = 60
I = 90
I = 150
0.1
0.2
v, mOD/sec
0.4
v / [NAD+]
0.2
v, mOD/sec
0.0005
0.0005
0.001
v / [NAD+]
0.0015
0.0005
v / [NAD+]
0.001
0.0015
Figure S10: Eadie-Hofstee plots corresponding to the results of fit in Figure S9. For details see
text.
be effectively indistinguishable from zero. In that case, the steady-state initial rate equation (in
the absence of added inhibitors) for BaIMPDHL would reduce to the algebraic form shown in
Eqns (S43)(S52). These equations were automatically derived by the DynaFit software package
using the following input text:
[mechanism]
enzyme E.S
reaction N ---> P + NH
E.S + N ---> E.S.N
E.S.N <==> E.P.NH
E.P.NH <==> E.P + NH
E.P ---> E.S + P
E.P + N <==> E.P.N
:
:
:
:
:
k1
k2
k3
k4
k5
26
k-2
k-3
k-5
v = [E]0
par/set
initial
1
2
3
4
5
6
Km / 1
Ki / 1
kK / 1
Km / 2
Ki / 2
kK / 2
500
5000
0.01
500
5000
0.01
n1 [N]
d1 + d2 [N] + d3 [N][NH] + d4 [N]2
(S43)
d1
= 1
(S44)
d2
k1 (k2 k4 + k3 k4 + k2 k4 + k2 k3 )
k2 k3 k4
(S45)
d3
k1 k3 (k2 + k2 )
k2 k3 k4
(S46)
d4
k1 k5
k4 k5
(S47)
kcat(f) =
n1
d2
k2 k3 k4
k2 k4 + k3 k4 + k2 k4 + k2 k3
(S48)
Km =
d1
d2
k2 k3 k4
k1 (k2 k4 + k3 k4 + k2 k4 + k2 k3 )
(S49)
kcat n1
=
Km d1
k1
(S50)
Ki(N) =
d2
d4
k5 (k2 k4 + k3 k4 + k2 k4 + k2 k3 )
k2 k3 k5
(S51)
Ki(NH,N) =
d2
d3
k2 k4 + k3 k4 + k2 k4 + k2 k3
k3 (k2 + k2 )
(S52)
final std.err.
500
7000
0.0247
1030
7800
0.0104
30
600
0.0008
90
900
0.0004
cv,%
low
high
6.0
7.9
3.4
8.4
11.0
4.2
440
5900
0.0229
870
6200
0.0095
560
8300
0.0265
1240
9800
0.0114
note
Table S7: Results of fit from the analysis of kinetic isotope effect. All experiments were performed in the same session (i.e., identical enzyme concentration) at saturating [IMP] = 1 mM.
The notation "/1" refers to results obtained with 1 H-IMP, whereas "/2" refers to results obtained
with 2 H-IMP. The optimized parameter kK is defined as the corresponding kcat /Km . Asymmetric
confidence interval bounds low and high are at the 95% confidence level.
Note that in this simplified kinetic mechanism the specificity constant kcat /Km is by definition
equal to the microscopic association rate constant k1 . More generally, kcat /Km values always
include only those microscopic rate constants up to (and including) the first irreversible step
27
1
H-IMP
2
0.2
0
v, mOD/sec
0.4
H-IMP
2000
4000
[NAD+], M
6000
(app)
8000
(app)
Figure S11: Determination of Km and kcat (assuming [E]0 = 10 nM) for NAD+ at saturating
concentration (1.0 mM) of either 1 H-IMP or 2 H-IMP, as indicated in the figure margin.
[22]. Therefore, if NAD+ binding were in fact sticky, then the experimentally observed kcat /Km
value should be entirely insensitive to isotopic substitution in 1 H-IMP vs. 2 H-IMP.
To test whether k1 is effectively zero or nonzero (as is suggested by the stopped-flow experiments), we measured kcat /Km either with 1 H-IMP or with 2 H-IMP at saturating concentrations.
The regression model was Eqn (S53), where vobs is the observed initial rate in mOD/sec; rQ is the
molar response coefficient of NADH (6.22 mOD/M); [E]0 is the active enzyme concentration
(10 nM); Km is the Michaelis constant; Ki is the substrate inhibition constant; and kK is kcat /Km
for the given isotope. Here we utilized Northrops [23] rearrangement of the Michaelis-Menten
equation (adjusted for the substrate inhibition term containing Ki ). The reason for recasting
the rate equation in this particular way is that, as was documented in ref. [23], the algebraic
rearrangement significantly increases the accuracy of specifically kcat /Km determinations. The
requisite DynaFit script is listed in Appendix A.2.3.
vobs
[S]0
Km + [S]0 + [S]20 /Ki
!
Km [S]0
kcat
Km Km + [S]0 + [S]20 /Ki
= rQ [E]0 kcat
= rQ [E]0
= rQ [E]0 kK
kK
kcat
Km
Km [S]0
Km + [S]0 (1 + [S]0 /Ki )
(S53)
(S54)
28
The results of fit are summarized graphically in Figure S11. The best-fit model parameter
values are listed in Table S7. Notation /1" and /2" refers to 1 H-IMP vs. 2 H-IMP, respectively. Columns low and high contain the 95% confidence intervals computed by the profilet method of Bates & Watts [9, 11]. The best-fit parameter values listed in Table S7 show that
for 1 H-IMP kK kcat /Km = (0.0247 0.0008) M1 s1 whereas for 2 H-IMP kK kcat /Km =
(0.0104 0.0004) M1 s1 . Thus the observed kinetic isotope effect is both surprisingly large
and also highly statistically significant. Note that the coefficient of variation for both 1 H-IMP and
2
H-IMP determination of kK is lower than 5%. This high level of precision would not be possible
to achieve with the classical as opposed to the rearranged [23] algebraic form of the MichaelisMenten equation. Using the standard formula for statistical uncertainty of a ratio from error
propagation theory [24, p. 62, Eqn 4-11], the most probable value and the standard error7 of the
kcat /Km isotope effect can be computed as
kcat
Km
!
=
!2
!2
0.0247
0.0008
0.0004
+
1
0.0104
0.0247
0.0104
= 2.38 0.12
Assuming that the intrinsic isotope effect on hydrogen transfer in either direction is equal to
approximately D k3 =D k3 = 4.0, the predicted value of the observed isotope effect on kcat /Km is
approximately D (kcat /Km ) = 1.34. This value was calculated using the microscopic rate constants
listed in Table S5 applied to the system of Eqns (S25)(S33). Thus the observed isotope effect
is even significantly larger than predicted, which provides further support to the conclusion that
NAD+ is not a sticky substrate for BaIMPDHL, in contrast with other IMPDH isoforms [5].
3.3.5. Double inhibition experiment
Experimental design and preliminary data
The results of both the stopped-flow kinetic investigations as well as steady-state initial rate
measurements described thus far clearly indicate that the inhibitor A110 binds strongly (Kd 50
nM) either to the covalent intermediate E-P, or to the noncovalent complex E.P, or to both of
these enzyme species. Significantly weaker binding (Kd 0.7 M) is observed to the enzyme
substrate complex E.A. Under IMP saturating conditions it is impossible to discriminate between
these alternate scenario or asses their relative contributions.
The experiments described and analyzed in this section were designed to decide whether or
not A110 binds preferentially either to E-P or to E.P. The experimental design relies on a doubleinhibitor assay involving A110 as the inhibitor of interest and XMP (P) as product inhibitor. The
experimental design for this semi-quantitative study was as follows:
7 Note that the covariance term in Bevingtons Eqn 4-11 can be neglected because the two determinations of k /K
cat
m
are statistically independent, having originated in separate experiments.
29
B
XMP : Ki(app) = (1160 +/- 50) M
0.2
0.4
v, mOD/sec
0.2
v, mOD/sec
0.6
0.4
0.8
500
1000
[IMP], M
1000
[XMP], M
(app)
(app)
Figure S12: A: Determination of Km(A) at [B] = 1.5 mM. B: Determination of Ki(P) at [A] =
200 M and [B] = 1.5 mM.
30
2000
Ki , M Std. Error
0.141
0.153
0.145
0.163
0.224
0.333
0.007
0.008
0.008
0.011
0.023
0.058
Table S8: Experimentally observed dependence of the apparent inhibition constant, Ki , for
A110 at [IMP] = 200 M and [NAD+ ] = 1.5 mM.
Note in Table S8 that the formal standard error of determination increases with increased
[XMP] concentration. This is a consequence of the fact that as the two highest values of [XMP]
the apparent inhibition constant (Ki > 200 nM) already increased above the highest concentration of A110 used in this experiment ([A110]max = 180 nM). If all inhibitor concentrations
utilized in a dose-response experiment are lower than the apparent inhibition constant (often
identical to the IC50 unless inhibitor depletion is involved), the precision and accuracy of Ki is
adversely affected.
Theoretical prediction for the dependence of Ki on [P]
With the preliminary data in hand regarding inhibitory potency of XMP and the apparent
Michaelis constant for IMP, it is possible set up a theoretical model for the double-inhibitor
experiment, by invoking the kinetic mechanism shown in Figure S13. Note that, for simplicity
in this semi-quantitative analysis, it was assumed that (i) the kinetic mechanism is effectively Bi
Bi Ordered and (ii) that the inhibitor does not bind to the free unliganded enzyme.
The microscopic rate constants k2 , k2 , k3 , k3 , k4 , k4 , k5 , k7 , k7 , k8 , k8 , k9 , and k9 were set
to the values determined in the stopped-flow kinetic experiment (Table S5). Approximate values
(app)
of the microscopic rate constants k1 , k1 were estimated on the basis of the Km(A) value shown in
Figure S12 (left panel). Similarly, approximate values of the microscopic rate constants k6 , k6
(app)
were estimated on the basis of the Ki(P) value shown in Figure S12 (right panel). The focus in
the heuristic simulation Ki vs. [P] is mainly on the plausible values of the equilibrium constant
k10 /k10 , measures the binding affinity (if any) of A110 toward the noncovalent complex E.P.
A DynaFit script containing the following input was utilized to derive steady-state initial rate
equation corresponding to the kinetic mechanism in Figure S13:
[task]
...
data = rates
approximation = king-altman
31
E-P.B
E.B
k7 k-7
+B
k3
E
E.A.B
k2
+A
k-1
k5
k4
E-P
E-P.Q
k-2
k-9
E.A
A
B
P
Q
I
+I
k-8
k8
IMP
NAD+
XMP
NADH
inhibitor
E
+P k-6
+I
+B
k1
H2O
+Q k-4
k-3
k6
E.P
+I
k9
k-10
k10
E.P.I
E-P.I
E.I
E.A.I
k1
k2
k3
k4
k5
k6
k7
k-1
k-2
k-3
k-4
k8
k9
k10
k-8
k-9
k-10
k-6
k-7
v =
[E]0
n1 [A][B]
D =
(S55)
(S56)
(S57)
k1 k2 k3 k4
k1 (k2 k3 + k2 k4 + k3 k4 )
n1
(S58)
d1
= 1
d2
k6
k6
(S60)
d3
k2 k3 k4
(k
k5 2 k3 + k2 k4 + k3 k4 )
(S61)
d4
k2 k3 k4
k1 (k2 k3 + k2 k4 + k3 k4 )
(S62)
d5
k1
k1
(S63)
d6
k6 k10
k6 k10
(S64)
d7
k2 k3 k4 k6
k5 k6 (k2 k3 + k2 k4 + k3 k4 )
(S65)
d8
k2 k3 k4 k6
k1 k6 (k2 k3 + k2 k4 + k3 k4 )
(S66)
d9
k1 k8
k1 k8
(S67)
d10
k1 k2 k3 k4
k1 k5 (k2 k3 + k2 k4 + k3 k4 )
(S68)
(S59)
d11
k1 k2 (k3 k5 k6 + k4 k5 k6 + k3 k5 k6 + k3 k4 k6 + k3 k4 k5 )
k1 k5 k6 (k2 k3 + k2 k4 + k3 k4 )
(S69)
d12
k2 k3 k4 k6 k10
k5 k6 k10 (k2 k3 + k2 k4 + k3 k4 )
(S70)
d13
k2 k3 k4 k6 k10
k1 k6 k10 (k2 k3 + k2 k4 + k3 k4 )
(S71)
d14
k1 k2 k3 k4 k8
k1 k5 k8 (k2 k3 + k2 k4 + k3 k4 )
(S72)
d15
(S73)
d16
k1 k2 k4 (k3 + k3 )
k1 k5 (k2 k3 + k2 k4 + k3 k4 )
(S74)
d17
k1 k2 k3 k4 k7
k1 k5 k7 (k2 k3 + k2 k4 + k3 k4 )
(S75)
kcat =
n1
d11
k3 k4 k5 k6
k3 k5 k6 + k4 k5 k6 + k3 k5 k6 + k3 k4 k6 + k3 k4 k5
(S76)
Km(A) =
d4
d11
k3 k4 k5 k6
k1 (k3 k5 k6 + k4 k5 k6 + k3 k5 k6 + k3 k4 k6 + k3 k4 k5 )
(S77)
Km(B) =
d5
d11
k5 k6 (k2 k3 + k2 k4 + k3 k4 )
k2 (k3 k5 k6 + k4 k5 k6 + k3 k5 k6 + k3 k4 k6 + k3 k4 k5 )
(S78)
n1
kcat
=
Km(A) d4
= k1
kcat
n1
=
Km(B) d5
(S79)
k2 k3 k4
k2 k3 + k2 k4 + k3 k4
34
(S80)
Ki(I,P) =
d2
d6
k10
k10
(S81)
Ki(I,A) =
d5
d9
k8
k8
(S82)
d11
d15
k9 k10 (k3 k5 k6 + k4 k5 k6 + k3 k5 k6 + k3 k4 k6 + k3 k4 k5 )
k3 k4 (k6 k9 k10 + k5 k9 k10 )
(S83)
Ki(A) =
d1
d5
k1
k1
(S84)
Ki(B) =
d1
d4
k1 (k2 k3 + k2 k4 + k3 k4 )
k2 k3 k4
(S85)
Ki(B,A,Q) =
d10
d16
k2 k3
k2 (k3 + k3 )
(S86)
Ki(B,A) =
d11
d17
k7 (k3 k5 k6 + k4 k5 k6 + k3 k5 k6 + k3 k4 k6 + k3 k4 k5 )
k3 k4 k6 k7
(S87)
d1
d3
k5 (k2 k3 + k2 k4 + k3 k4 )
k2 k3 k4
(S88)
d11
d16
k3 k5 k6 + k4 k5 k6 + k3 k5 k6 + k3 k4 k6 + k3 k4 k5
k4 k6 (k3 + k3 )
(S89)
d1
d2
k6
k6
(S90)
Ki(I,A,B) =
Ki(Q) =
Ki(Q,A,B) =
Ki(P) =
v =
[E]
kcat(f)
35
[A][B]
Ki(B) Km(A)
(S91)
(S92)
D =
1+
[Q]
[B]
[A]
[P]
+
+
+
Ki(P) Ki(Q) Ki(B) Ki(A)
[P][I]
[Q][P]
[B][P]
[A][I]
+
+
+
Ki(P) Ki(I,P) Ki(Q) Ki(P) Ki(B) Ki(P) Ki(A) Ki(I,A)
[A][Q]
[A][B]
[Q][P][I]
+
+
Ki(A) Ki(Q) Ki(B) Km(A) Ki(Q) Ki(P) Ki(I,P)
[B][P][I]
[A][Q][I]
[A][B][I]
+
+
Ki(B) Ki(P) Ki(I,P) Ki(A) Ki(Q) Ki(I,A) Ki(B) Km(A) Ki(I,A,B)
[A][B][Q]
[A][B]2
+
Ki(B) Km(A) Ki(Q,A,B) Ki(B) Km(A) Ki(B,A)
(S93)
D0
[A]
[Q][P]
[P]
+
+
Ki(P) Ki(I,P) Ki(A) Ki(I,A) Ki(Q) Ki(P) Ki(I,P)
+
D0
(S94)
where
[B][P]
[A][Q]
[A][B]
+
+
Ki(B) Ki(P) Ki(I,P) Ki(A) Ki(Q) Ki(I,A) Ki(B) Km(A) Ki(I,A,B)
= 1+
(S95)
[P]
[Q]
[B]
[A]
+
+
+
Ki(P) Ki(Q) Ki(B) Ki(A)
[Q][P]
[B][P]
[A][Q]
+
+
Ki(Q) Ki(P) Ki(B) Ki(P) Ki(A) Ki(Q)
[A][B]
[A][B][Q]
[A][B]2
+
+
Ki(B) Km(A) Ki(B) Km(A) Ki(Q,A,B) Ki(B) Km(A) Ki(B,A)
(S96)
In the special case of an experiment where NADH is absent in the assay mixture, algebraic
terms with [Q] vanish from the definition of the apparent inhibition constant, in which case D0
and simplify as follows:
36
D0
1+
[P]
[B]
[A]
[B][P]
+
+
+
Ki(P) Ki(B) Ki(A) Ki(B) Ki(P)
[A][B]
[A][B]2
+
Ki(B) Km(A) Ki(B) Km(A) Ki(B,A)
(S97)
[P]
[A]
+
Ki(P) Ki(I,P) Ki(A) Ki(I,A)
+
[A][B]
[B][P]
+
Ki(B) Ki(P) Ki(I,P) Ki(B) Km(A) Ki(I,A,B)
(S98)
Heuristic simulations
Based on the kinetic scheme in Figure S13, we have simulated a collection of theoretical
curves representing the dependence of the apparent inhibition constant Ki on the presumed relative binding affinities of I toward the covalent intermediate and toward the noncovalent complex,
assuming that both types of binding phenomena can occur at the same time. The requisite DynaFit simulation script is shown in Appendix A.2.7. We assumed that Ki(I,A) was 0.7 M, Ki(I,A,B)
was 60 nM, thus assuming that I binds to covalent intermediate with high affinity. The inhibition
constant Ki(I,P) was varied from 10 nM to 5.12 M. The results are shown in Figure S14.
Ki(I,A) = 0.7, Ki(I,A,B) = 0.06 M
0.2
Ki(I,P) = 1.24
Ki(I,P) = 0.64
Ki(I,P) = 0.32
, M
Ki(I,P) = 0.08
Ki(I,P) = 0.04 uM
0.15
0.1
0.1
Ki
(app)
0.2
values
Ki(I,P) = 1.24
Ki(I,P) = 0.64
Ki(I,P) = 0.32
Ki(I,P) = 0.02
Ki(I,P) = 0.01 uM
Ki
(app)
Ki(I,P) = 5.12
Ki(I,P) = 2.56
Ki(I,P) = 0.16
Ki(I,P) = 0.08
Ki(I,P) = 0.04
(app)
, M
0.3
1000
2000
[P], M
(a)
500
1000
[XMP], M
(b)
Figure S14: (a) Simulation of Ki values in dependence on the concentrations of [P]. (b) Linear
least squares fit of simulated dependence of Ki vs. [P]. The assumed true value of Ki(I,A,B) ,
which measures inhibitor binding to the covalent intermediate E-P, was 60 nM throughout the
simulation. The assumed true value of Ki(I) , which measures inhibitor binding to the noncovalent complex EP varied from 40 nM to 5.12 M and is shown in the margin.
The results show that the dependence of Ki on [P] is complex. The plots have various shapes
37
Ki(I,P) , M
5.12
2.56
1.28
0.64
experiment
0.32
0.16
0.1
0.1
0.1
0.1
0.6
0.1
0.1
Table S9: Comparison of experimentally observed slope of the plot of Ki vs. [P] (Figure S14B)
with the slopes simulated at various assumed values of the inhibition constant Ki(I,P) .
at least ten-fold higher than the inhibition constant Ki(I,A,B) = 60 nM, which measures the the
binding affinity of A110 toward the covalent intermediate.
3.4. Steady-state kinetics: Summary and conclusions
Based on the results of the combined initial-rate experiments, we can arrive at the following
mechanistic conclusions:
1. Under IMP saturating conditions, the uncompetitive inhibition constant of A110, defined
in terms of microscopic rate constants by Eqn (S29), is Ki(I,B) 50 nM. This suggests
strong binding to the covalent intermediate E-P or to the noncovalent enzymeproduct
complex E.P.
2. The double-inhibitor experiment conducted under IMP sub-saturation suggests that the
inhibitor binds preferentially to the covalent intermediate. A semi-quantitative assessment
suggest at least ten-fold stronger binding, as measured by the inhibition constant.
3. The uncompetitive inhibition constant Ki(I,B) is not by definition equal to the dissociation
complex of the requisite ternary complex, contrary to generalized suggestions in textbook
literature regarding the nature of steady-state inhibition constants.
4. The inhibitor also binds relatively weakly (Ki(I) = Kd(EA,I) 0.7 M) to the enzyme
substrate (IMP) complex. In this case the inhibition and dissociation constants are by
definition identical.
5. Thus, under IMP saturating conditions, in the conventional nomenclature [25] A110 would
be characterized as a mixed-type, predominantly uncompetitive inhibitor.
6. The substrate kinetic properties of BaIMPDHL are well reproduced in the stopped-flow
vs. initial rate data. This includes quantitative measures of (a) the saturation behavior; (b)
substrate inhibition by NAD+ ; and (c) product inhibition by NADH.
4. Responses to Reviewers
4.1. Linear vs. logarithmic plots
A Reviewer has raised a question regarding the relative merits of linearly scaled vs. logarithmically scaled time axis, in plotting stopped-flow transient kinetic data. One particular
suggestion by the Reviewer was that perhaps plotting stopped-flow data in linear as opposed to
logarithmic time increases our ability to detect any possible lag phase transients.
39
This gives us an opportunity to demonstrate that lag type transients are extremely difficult
to detect by naked eye simply by examining the plots of experimental data and the overlayed
model curves, regardless of which type of scaling (linear or logarithmic) is used. However, an
exquisitely informative alternative is presented by plotting instantaneous reaction rates vs. time,
again regardless of which type of scaling is used on the time axis.
[NADH] = 0, [A110] = 0
400
d(1000 A340) / dt
200
0
0.5
0
-0.5
residuals
0.25
0.5
1
2
4
6
8 mM
600
40
[NADH] = 0, [A110] = 0
20
1000 A340
60
80
0.25
0.5
1
2
4
6
8 mM
0.2
0.4
0.2
0.4
time, s
time, s
(a)
(b)
(c)
(d)
Figure S15: (a) The upper-left panel in Figure S2 above, redrawn in linear time scale. (b)
Corresponding instantaneous rate plots. (c) Magnified portion of the lowest concentration curve,
[NAD] = 0.25 mM, showing the slight lag transient. (d) Corresponding instantaneous rate plot.
Figure S15a displays what was shown previously as the upper-left panel in Figure S2 above,
this time with linearly scaled time. Figure S15b shows the corresponding instantaneous rate
plots. Recall that, at an arbitrary reaction time t, the instantaneous rate is the slope of a tangent
40
line drawn with respect to the corresponding reaction progress curve. Most importantly, any
observed increase in the instantaneous rate curve represent a lag phase being present in the
underlying theoretical model curve.
Please note that only an extremely well-trained eye could possibly detect a very slight lag,
occurring with the first approximately 50 milliseconds especially in the low-concentration progress
curves shown Figure S15a. Now note that this lag is rendered prominently visible in the accompanying instantaneous rate plots, Figure S15b. All instantaneous rate curves in Figure S15b
display an increase in the reaction rate between time t = 0 and t = 30 msec.
A visual verification of the very subtle but statistically significant lag is presented in Figure
S15c and Figure S15d. The lag phase becomes visually detectable in the data plot (as opposed
to the instantaneous rate plot) only by zooming very purposely on the initial approximately
10% section (the first 50 msec of the total 500 msec trace). In contrast, the instantaneous rate
plot shows a very clear lag (i.e., increase in the observed instantaneous rate) for all progress
curves plotted in their entirety.
[NADH] = 0, [A110] = 6 M
[NADH] = 0, [A110] = 6 M
d(1000 A340) / dt
400
600
0.25
0.5
1
2
4
6
8 mM
200
20
0
1000 A340
40
0.25
0.5
1
2
4
6
8 mM
0
-1
residuals
0.5
-0.5
-1.5
time, s
time, s
(a)
(b)
Figure S16: (a) The lower-left panel in Figure S2 above, redrawn in linear time scale. (b)
Corresponding instantaneous rate plots.
This Reviewer also suggested that the shoulder feature between t = 0.1 sec and t = 0.5
sec, displayed in the lower left panel of Figure S2 and discussed in section 2.3.1, could be easily
visualized as a simple approach to steady-state, if plotted in the linear as opposed to logarithmic
time coordinates. Figure S16 helps explain that this statement is only partially true. As a matter
of fact, several curves shown in Figure S16 (in particular [NAD] = 0.25 and 0.5 mM) never
actually reach steady-state. Note that the [NAD] = 0.25 and 0.5 mM curves never develop a
truly linear phase corresponding to genuine steady-state. The reason is that in the presence of
the inhibitor at [I] = 6 M there exists a complex interplay between a large number competing
processes, as befits a reaction mechanism involving thirteen microscopic steps.
41
References
[1] P. Kuzmic, Program DYNAFIT for the analysis of enzyme kinetic data: Application to HIV
proteinase, Anal. Biochem. 237 (1996) 260273.
[2] P. Kuzmic, DynaFit - A software package for enzymology, Meth. Enzymol. 467 (2009)
247280.
[3] J. M. Beechem, Global analysis of biochemical and biophysical data, Meth. Enzymol. 210
(1992) 3754.
[4] A. Raue, C. Kreutz, T. Maiwald, J. Bachmann, M. Schilling, U. Klingmller, J. Timmer,
Structural and practical identifiability analysis of partially observed dynamical models by
exploiting the profile likelihood, Bioinformatics 25 (2009) 19231929.
[5] T. V. Riera, W. Wang, H. R. Josephine, L. Hedstrom, A kinetic alignment of orthologous
inosine-5-monophosphate dehydrogenases, Biochemistry 47 (2008) 86898696.
[6] L. Hedstrom, IMP dehydrogenase: structure, mechanism, and inhibition, Chem. Rev. 109
(2009) 29032928.
[7] K. V. Price, R. M. Storm, J. A. Lampinen, Differential Evolution - A Practical Approach to
Global Optimization, Springer Verlag, Berlin - Heidelberg, 2005.
[8] M. L. Johnson, Use of least-squares techniques in biochemistry, Meth. Enzymol. 240
(1994) 122.
[9] D. M. Bates, D. G. Watts, Nonlinear Regression Analysis and its Applications, Wiley, New
York, 1988.
[10] I. Brooks, D. Watts, K. Soneson, P. Hensley, Determining confidence intervals for parameters derived from analysis of equilibrium analytical ultracentrifugation data, Meth. Enzymol. 240 (1994) 45978.
[11] D. G. Watts, Parameter estimation from nonlinear models, Methods Enzymol. 240 (1994)
2436.
[12] K. A. Johnson, Z. B. Simpson, T. Blom, Global Kinetic Explorer: A new computer program
for dynamic simulation and fitting of kinetic data, Anal. Biochem. 387 (2009) 2029.
[13] K. A. Johnson, Z. B. Simpson, T. Blom, FitSpace Explorer: An algorithm to evaluate
multidimensional parameter space in fitting kinetic data, Anal. Biochem. 387 (2009) 3041.
[14] K. A. Johnson, Fitting enzyme kinetic data with KinTek Global Kinetic Explorer, Meth.
Enzymol. 267 (2009) 601626.
[15] H. Bisswanger, Enzyme Kinetics, 2nd Edition, Wiley-VCH, Tuebingen, 2008.
[16] I. H. Segel, Enzyme Kinetics, Wiley, New York, 1975.
[17] J. F. Morrison, Kinetics of the reversible inhibition of enzyme-catalysed reactions by tightbinding inhibitors, Biochim. Biophys. Acta 185 (1969) 269286.
42
[18] E. L. King, C. Altman, A schematic method of deriving the rate laws for enzyme-catalyzed
reactions, J. Phys. Chem. 60 (1956) 13751378.
[19] W. Cleland, The kinetics of enzyme-catalyzed reactions with two or more substrates or
products: I. nomenclature and rate equations, Biochim. Biophys. Acta 67 (1963) 104137.
[20] S. Cha, Tight-binding inhibitors. i. kinetic behavior, Biochem. Pharmacol. 24 (1975) 2177
2185.
[21] J. W. Williams, J. F. Morrison, The kinetics of reversible tight-binding inhibition, Meth.
Enzymol. 63 (1979) 437467.
[22] D. H. Rich, D. B. Northrop, Enzyme kinetics in drug design: Implications of multiple forms
of enzyme on substrate and inhibitor structure-activity correlations, in: T. J. Perun, C. L.
Prost (Eds.), Computer-Aided Drug Design: Methods and Applications, Marcel Dekker,
New York, 1989, pp. 185250.
[23] D. Northrop, Fitting enzyme-kinetic data to v/k, Anal. Biochem. 321 (1983) 457461.
[24] P. R. Bevington, Data Reduction and Error Analysis in the Physical Sciences, McGraw-Hill,
New York, 1969.
[25] International Union of Biochemistry (IUB), Symbolism and terminology in enzyme kinetics, Biochem. J. 213 (1981) 561571.
43
Appendix
A. DynaFit scripts
A.1. Global fit of stopped-flow data
This DynaFit [2] script performs global fit of combined transient kinetic data using one of the
16 combinatorial replicates (replicate no. 1 listed in Table S2). The double question marks (??)
identify the model parameters to be subjected to the full asymmetric confidence interval search.
[task]
task = fit
data = progress
[mechanism]
E.A + B <==> E.A.B
:
k2
k-2
E.A.B <==> E.P.Q
:
k3
k-3
E.P.Q <==> E.P + Q
:
k4
k-4
E.P ---> E.A + P
:
k5
E.P + B <==> E.P.B
:
k7
k-7
E.A + I <==> E.A.I
:
k8
k-8
E.P + I <==> E.P.I
:
k9
k-9
[constants]
k2 = 0.01 ?? ,
k-2 = 10 ??
k3 = 100 ??
,
k-3 = 10 ??
k4 = 1000 ?? ,
k-4 = 10 ??
k5 = 10 ??
k7 = 0.01 ?? ,
k-7 = 100 ??
k8 = 10 ??
,
k-8 = 10 ??
k9 = 1 ??
,
k-9 = 0.1 ??
[parameters]
In = 6 ?
[concentrations]
E.A = 2.5 ?
[responses]
Q = 6.22
E.P.Q = 1 * Q
[data]
directory ./project/impdh/01/progress/data
monitor
E.A, E.A.B, E.P.Q, E.P, E.P.B, E.A.I, E.P.I
plot
logarithmic
;
; Control experiment -- DMSO only:
;
graph [NADH] = 0, [A110] = 0
maximum
0.5
sheet
N1.csv
shift 0 | column 2 | offset auto ? | conc B = 250 |
shift 1 | column 3 | offset auto ? | conc B = 500 |
shift 2 | column 4 | offset auto ? | conc B = 1000 |
shift 4 | column 5 | offset auto ? | conc B = 2000 |
44
label
label
label
label
0.25
0.5
1
2
45
46
sheet nad.csv
column 3 | conc E.A = 1 * cE1 | label A = 1000 (b)
column 2 | conc E.A = 1 * cE2 | label A = 1000 (c)
graph NAD + NADH
sheet nad-nadh.csv
column 2 | conc E.A = 1 * cE3, Q =
0 | label Q =
column 3 | conc E.A = 1 * cE3, Q = 50 | label Q =
column 4 | conc E.A = 1 * cE3, Q = 100 | label Q =
column 5 | conc E.A = 1 * cE3, Q = 150 | label Q =
graph NAD (low) + A110
sheet nad-a110-d.csv
column 2 | conc E.A = 1 * cE4, I = 0.000 | label I
column 3 | conc E.A = 1 * cE4, I = 0.015 | label I
column 4 | conc E.A = 1 * cE4, I = 0.030 | label I
column 5 | conc E.A = 1 * cE4, I = 0.060 | label I
column 6 | conc E.A = 1 * cE4, I = 0.120 | label I
column 7 | conc E.A = 1 * cE4, I = 0.180 | label I
graph NAD (high) + A110
sheet nad-a110-a.csv
column 2 | conc E.A = 1 * cE5, I = 0.000 | label I
column 3 | conc E.A = 1 * cE5, I = 0.015 | label I
column 4 | conc E.A = 1 * cE5, I = 0.030 | label I
column 5 | conc E.A = 1 * cE5, I = 0.060 | label I
column 6 | conc E.A = 1 * cE5, I = 0.090 | label I
column 7 | conc E.A = 1 * cE5, I = 0.180 | label I
[output]
directory ./project/IMPDH/01/rates/output/fit-micro
[settings]
{Filter}
XMax = 7000
{Output}
WriteEPS = y
WriteTeX = y
ResidualsEPS = n
XAxisLabel = [NAD^+], {/Symbol m}M
YAxisLabel = v, mOD/min
[end]
0 uM
50
100
150
=
=
=
=
=
=
0 uM
0.015
0.030
0.060
0.120
0.180
=
=
=
=
=
=
0 uM
0.015
0.030
0.060
0.090
0.150
47
data = generic
[parameters]
E, B, Q, I
kcat, KmB, KiI, KiIB, KiB, KiQ, KiQB
rQ
cE1, cE2, cE3, cE4, cE5
[model]
;
; Enzyme concentrations in different experiments: All optimized.
;
cE1 = 0.01 ?
cE2 = 0.01 ?
cE3 = 0.01 ?
cE4 = 0.01 ?
cE5 = 0.01 ?
;
; Extinction coefficient of NADH:
;
rQ = 6.22
; fixed !
;
; Predicted kinetic constants: All fixed in the regression!
;
kcat = 11.6
KmB = 415
KiI = 0.791
KiIB = 0.0572
KiB = 6610
KiQ = 301
KiQB = 87.2
;
; "Morrison equation" (uM/sec) for mechanism at saturating [IMP]:
;
n = B/KmB
d = 1 + Q/KiQ + B/KmB*(1 + Q/KiQB + B/KiB)
alpha = 1/KiI*(1 + Q/KiQ) + B/(KmB*KiIB)
Kiapp = d/alpha
e = E - I - Kiapp
v = kcat * n/d * (e + sqrt (e*e + 4*Kiapp*E))/2
;
; Observed rate (mOD/sec):
;
vObs = v * rQ
[data]
variable B
directory ./project/IMPDH/01/rates/data
graph NAD
sheet nad-b.csv
column 2 | param E = 1 * cE1 | label A = 0.75 mM
column 3 | param E = 1 * cE1 | label A = 1.0 (a)
sheet nad.csv
column 3 | param E = 1 * cE1 | label A = 1.0 (b)
48
49
S
Km, Ki, kK, Eo, rP
[model]
rP = 6.22
Eo = 0.010
v = rP * Eo * kK * Km * S/(Km + S*(1 + S/Ki))
[data]
variable S
directory ./project/impdh/01/rates/data
sheet nad-isotope.csv
column 2 | label ^{1}H-IMP
param Km = 500 ??, Ki = 5000 ??, kK = 0.01 ??
column 3 | label ^{2}H-IMP
param Km = 500 ??, Ki = 5000 ??, kK = 0.01 ??
[output]
directory ./project/impdh/01/rates/output/fit-isotope
[settings]
{Output}
WriteEPS = yes
ResidualsEPS = no
WriteTeX = yes
XAxisLabel = [NAD^+], {/Symbol m}M
YAxisLabel = v, mOD/sec
[end]
50
YAxisLabel = v, mOD/sec
ResidualsEPS = n
WriteTeX = y
[set:rates]
IMP rate
10 0.0736
20 0.1308
30 0.1702
50 0.2327
75 0.2593
100 0.3098
150 0.3617
200 0.3837
300 0.4192
500 0.4493
750 0.4545
1000 0.4863
[end]
51
125 0.8009
250 0.7491
500 0.6407
1000 0.4744
2000 0.3232
[end]
0 uM
125
250
500
1000
2000
52
[parameters]
A, B, P
; concentrations
KmA, KiB, KiP, KiBA
; substrate kinetic constants
KiIA, KiIP, KiIAB
; inhibition constants for "I"
[model]
A = 200
; concentrations (P: see [data])
B = 1500
KmA = 50
; substrate kinetic constants
KiA = 140
KiB = 1300
KiP = 220
KiBA = 6500
KiIA = 0.7
; inhibition constants (KiP: see [data])
KiIAB = 0.06
Do = 1 + P/KiP + B/KiB + A/KiA + B*P/(KiB*KiP) + A*B/(KiB*KmA)*(1 + B/KiBA)
g1 = P/(KiP*KiIP) + A/(KiA*KiIA)
g2 = B*P/(KiB*KiP*KiIP) + A*B/(KiB*KmA*KiIAB)
gamma = g1 + g2
KiApp = Do / gamma
[data]
variable P
mesh logarithmic from 2000 to 10 step 0.5
directory ./project/impdh/kiapp-xmp/data
graph K_{i(I,A)} = 0.7, K_{i(I,A,B)} = 0.06 {/Symbol m}M
sheet sim-002.csv
column 2 | param KiIP = 5.12 | label Ki(I,P) = 5.12
column 3 | param KiIP = 2.56 | label Ki(I,P) = 2.56
column 4 | param KiIP = 1.24 | label Ki(I,P) = 1.24
column 5 | param KiIP = 0.64 | label Ki(I,P) = 0.64
column 6 | param KiIP = 0.32 | label Ki(I,P) = 0.32
column 7 | param KiIP = 0.16 | label Ki(I,P) = 0.16
column 8 | param KiIP = 0.08 | label Ki(I,P) = 0.08
column 9 | param KiIP = 0.04 | label Ki(I,P) = 0.04
column 10 | param KiIP = 0.02 | label Ki(I,P) = 0.02
column 11 | param KiIP = 0.01 | label Ki(I,P) = 0.01 uM
[output]
directory ./project/impdh/kiapp-xmp/output/sim-002
[settings]
{Output}
XAxisLabel = [P], {/Symbol m}M
YAxisLabel = K_i^{(app)}, {/Symbol m}M
ResidualsEPS = n
[end]
53
task = fit
data = generic
[parameters]
XMP, Ki0, a
[model]
KiApp = Ki0 + XMP * a
[data]
directory ./project/impdh/kiapp-xmp/data
variable XMP
sheet sim-002b.csv
graph Linear fit of simulated K_{i}^{(app)} values
column 2 | param Ki0 = 0.1 ?, a = 0.0001 ? | label Ki(I,P)
column 3 | param Ki0 = 0.1 ?, a = 0.0001 ? | label Ki(I,P)
column 4 | param Ki0 = 0.1 ?, a = 0.0001 ? | label Ki(I,P)
column 5 | param Ki0 = 0.1 ?, a = 0.0001 ? | label Ki(I,P)
column 6 | param Ki0 = 0.1 ?, a = 0.0001 ? | label Ki(I,P)
column 7 | param Ki0 = 0.1 ?, a = 0.0001 ? | label Ki(I,P)
column 8 | param Ki0 = 0.1 ?, a = -0.0001 ? | label Ki(I,P)
[output]
directory ./project/impdh/01/rates/output/fit-kiapp-003
[settings]
{Filter}
XMax = 1000
{Output}
XAxisLabel = [XMP], {/Symbol m}M
YAxisLabel = K_i^{(app)}, {/Symbol m}M
ResidualsEPS = n
[end]
=
=
=
=
=
=
=
5.12
2.56
1.24
0.64
0.32
0.08
0.04 uM
54
[output]
directory ./project/impdh/01/rates/output/fit-kiapp-001
[settings]
{Output}
XAxisLabel = [XMP], {/Symbol m}M
YAxisLabel = K_i^{(app)}, {/Symbol m}M
IncludeYZero = y
ResidualsEPS = n
[end]
55
+---manual
|
+---project
<=== inserted directory
|
\---IMPDH
|
\---01
|
+---progress
|
|
\---data
|
\---rates
|
\---data
\---system
To reproduce any particular kinetic analysis reported here, please follow these steps:
1.
2.
3.
4.
Figure
Table
1, 2
1
3
2
4
S2
S4
S3 S5
S6
S5
S6
S9
S10
S11
S12
S14
S7
S9
(a)
(b)
Directory
./progress/fit
./progress/rapid
./rates
./rates
./rates
./progress/confid
./progress/confid
./progress/confid
./progress/confid
./progress/rapid
./rates
./rates
./rates
./rates
./rates
./rates
./rates
N1H1I1
NxHyIz (b)
fig-3-classic, fig-3-tight
fig-S9
fig-4A, fig-4B
N1H1I1
N1H1I1
N1H1I1
NxHyIz (b)
NxHyIz (b)
fig-3-classic, fig-3-tight
fig-S9
fig-S10
fig-S11
fig-S12A, fig-S12B
fig-S14A, fig-S14B
fig-S14B
Extension .TXT
16 Combinations: x=1-4, y=1,2, z=1,2
Table S10: Organization of DynaFit script files in the distributed ZIP archive file.
3. Double-click on the corresponding batch file.
Beware that the execution time will vary greatly. For example, most initial-rate analyses will
take only a few seconds to complete. In contrast, each individual script located in the directory
./progress/confid (confidence interval estimation for microscopic rate constants) will take approximately three and a half hours to complete. For this particular group of scripts, it is highly
advantageous to utilize a multi-processor, multi-core machine and process at least eight DynaFit
scripts in parallel. Under those conditions, the total computation time for the group of 16 combinatorial replicates in directory ./progress/confid is approximately seven hours on a 2.4 GHz
four-core hyper-threaded microprocessor (e.g., Intel i7).
B.2.2. Raw experimental data files
Table S11 lists all raw experimental data files (type .CSV, Comma-Separate Values) utilized
in this report. The files can be used to replicate all kinetic analyses reported here by using
alternate software packages and data-analysis methods.
57
Figure
Table
1, 2
1
3
2
4
S2
S4
S3 S5
S6
S5
S6
S9
S10
S11
S12
S14A
S14B
S7
S8
S9
(a)
(b)
(c)
Directory
./progress/data
./progress/data
./rates/data
./rates/data
./rates/data
./progress/data
./progress/data
./progress/data
./progress/data
./progress/data
./rates/data
./rates/data
./rates/data
./rates/data
./rates/data
./rates/data/simulated
./rates/data/simulated
./rates/data
N1H1I1
NxHyIz (b)
nad-b, nad, nad-nadh, nad-a110-d, nad-a110-a
nad-b, nad, nad-nadh, nad-a110-d, nad-a110-a
a110-xmp
N1H1I1
N1H1I1
N1H1I1
NxHyIz (b)
NxHyIz (b)
nad-b, nad, nad-nadh, nad-a110-d, nad-a110-a
nad-b, nad, nad-nadh, nad-a110-d, nad-a110-a
nad-b, nad, nad-nadh, nad-a110-d, nad-a110-a
nad-isotope
(c)
kiapp-vs-xmp
kiapp-vs-xmp
kiapp-vs-xmp
Extension .CSV
16 Combinations: x=1-4, y=1,2, z=1,2
Embedded data
Table S11: Organization of DynaFit script files in the distributed ZIP archive file.
58