Inhibition of Inosine-5 - Monophosphate Dehydrogenase From Bacillus Anthracis: Mechanism Revealed by Pre-Steady-State Kinetics

Article
pubs.acs.org/biochemistry
Inhibition of Inosine-5-monophosphate Dehydrogenase from

Bacillus anthracis: Mechanism Revealed by Pre-Steady-State Kinetics
Yang Wei,, Petr Kuzmic,, Runhan Yu, Gyan Modi,, and Lizbeth Hedstrom*,,
Department of Biology, Brandeis University, Waltham, Massachusetts 02454, United States

BioKin Ltd., Watertown, Massachusetts 02472, United States
Department of Chemistry, Brandeis University, Waltham, Massachusetts 02454, United States
S Supporting Information
*
ABSTRACT: Inosine-5-monophosphate dehydrogenase

(IMPDH) catalyzes the conversion of inosine 5-monophosphate (IMP) to xanthosine 5-monophosphate (XMP).
The enzyme is an emerging target for antimicrobial therapy.
The small molecule inhibitor A110 has been identied as a
potent and selective inhibitor of IMPDHs from a variety of
pathogenic microorganisms. A recent X-ray crystallographic
study reported that the inhibitor binds to the NAD+ cofactor
site and forms a ternary complex with IMP. Here we report a
pre-steady-state stopped-ow kinetic investigation of IMPDH from Bacillus anthracis designed to assess the kinetic signicance of
the crystallographic results. Stopped-ow kinetic experiments dened nine microscopic rate constants and two equilibrium
constants that characterize both the catalytic cycle and details of the inhibition mechanism. In combination with steady-state
initial rate studies, the results show that the inhibitor binds with high anity (Kd 50 nM) predominantly to the covalent
intermediate on the reaction pathway. Only a weak binding interaction (Kd 1 M) is observed between the inhibitor and E
IMP. Thus, the EIMPA110 ternary complex, observed by X-ray crystallography, is largely kinetically irrelevant.
of these compounds are also eective against IMPDHs from

bacteria such as B. anthracis and Mycobacterium tuberculosis,
suggesting their potential use as antibiotic agents.5,6,13
The triazole compound A110, which inhibits BaIMPDH
with an IC50 of 43 3 nM,15 displays in vitro antibacterial
activity against B. anthracis and Staphylococcus aureus.13 Related
A series compounds are uncompetitive inhibitors with respect
to IMP and noncompetitive (mixed) inhibitors with respect to
NAD+. These observations suggest that A110 binds after IMP
but do not reveal whether it binds to EIMP or another
downstream complex. Crystal structures of small molecule
inhibitor complexes with BaIMPDH and Clostridium perf ringes
IMPDH have been determined, indicating that A110 binds to
the cofactor site. However, these structures may not represent
the highest-anity enzymeinhibitor complexes.9,15,16
In this report, we utilize stopped-ow rapid kinetics
techniques, supported by conrmatory initial rate experiments,
to investigate the detailed inhibition mechanism of the small
molecule inhibitor A110 against a subdomain-deleted form of
the B. anthracis enzyme, BaIMPDHL.15 The results show that
A110 binds predominantly to the covalent intermediate. We
discuss these kinetic results in terms of possible conformational
and structural eects. We also report the values of microscopic
rate constants that characterize the catalytic cycle of
ntibiotic resistance is a worldwide problem threatening the

eective treatment of infections caused by pathogenic
bacteria.1,2 New antibiotics and targets are urgently needed.3 It
is also important to develop new antibiotics against potential
bioterrorism agents,4 such as Bacillus anthracis, the causative
agent of anthrax.
Inosine-5-monophosphate dehydrogenase (IMPDH) has
recently emerged as a promising antimicrobial drug target.5,6
IMPDH catalyzes the oxidation of inosine 5-monophosphate
(IMP) to xanthosine 5-monophosphate (XMP) with simultaneous reduction of the cofactor NAD+ to NADH.7 The
conversion of IMP to XMP is the rst and rate-limiting step in
the guanine nucleotide biosynthesis pathway. Inhibiting
IMPDH causes an imbalance in the purine nucleotide pool
that suppresses proliferation. The reaction involves the initial
attack of the active site Cys on C2 of IMP coupled to NAD+
reduction, to form a covalent intermediate. NADH departs; a
disordered ap folds into the empty cofactor site, and the
covalent intermediate undergoes hydrolysis. Several inhibitors
of mammalian IMPDHs, most notably mycophenolic acid, bind
selectively to the covalent intermediate and prevent hydrolysis.8
Selective inhibitors of IMPDH from Cryptosporidium parvum
were identied by high-throughput screening followed by
structural renement.915 These compounds eectively inhibit
CpIMPDH activity in vitro, display signicant antiparasitic
activity against an engineered Toxoplasma gondii strain relying
solely on CpIMPDH, and show a therapeutic eect in a mouse
model mimicking acute human cryptosporidiosis.912,14 Some
XXXX American Chemical Society
Received: March 23, 2016

Revised: August 17, 2016
DOI: 10.1021/acs.biochem.6b00265
Biochemistry XXXX, XXX, XXXXXX
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Biochemistry
BaIMPDHL. The mechanistic insights should prove to be
useful in the rational design of IMPDH-targeted therapy.
the adjustable baseline oset at 0.004 s, essentially a property of

the instrument, rQ (=6.22 mOD/M) is the molar response
coecient of NADH, [Q] is the concentration of NADH at
time t, and [EPQ] is the concentration of the E-PQ ternary
complex. It was assumed that the UV/vis extinction coecient
of NADH (6.22 103 mM1 cm1)20 does not change upon
binding to the enzyme, and therefore, the extinction coecients
of NADH and the enzyme complex are exactly identical.
The concentrations of both UV/vis detectable molecular
species ([Q] and [EPQ]) at time t were computed from their
initial concentrations at time zero by numerically solving an
initial value problem dened by a system of dierential
equations (S1S12 in the Supporting Information). The
numerical solution algorithm was the Livermore Solver of
ODE Systems (LSODE).21,22 The absolute global truncation
error tolerance was 1014 M; the relative global truncation
error was 108 (eight signicant digits).
For further details regarding data handling and analysis, see
the Supporting Information.
Steady-State Initial Rate Kinetics. Steady-state kinetic
experiments were performed by measuring initial velocities at
varying concentrations of NAD+, NADH, and A110 by
monitoring the production of NADH by the absorbance at
340 nm ( = 6.22 mM1 cm1) using a Hitachi U-2000 or
Shimadzu UV-2600 spectrophotometer. All measurements
were performed in the assay buer [50 mM Tris, 150 mM
KCl, and 1 mM DTT (pH 8.0)] at 25 C with a saturating
concentration of IMP (1 mM) and 14 nM enzyme (nominal
concentration) in a total assay volume of 1 mL in 1 cm path
length cuvettes. Initial rates were determined by either a linear
or an exponential t of the rst 5 min of the assay (17 time
points stepping by 20 s).
Steady-state initial rate data, obtained while simultaneously
varying the concentrations of NAD+, NADH, and A110, were
combined into a single multidimensional data set and subjected
to global regression analysis.19 The tting model is represented
by eq 2
MATERIALS AND METHODS

Materials. IMP disodium salt was purchased from MP
Biomedicals. NAD+ free acid was purchased from Roche, and
NADH disodium salt was purchased from Acros. Compound
A110 was synthesized as described previously.17 DTT, TCEP,
and IPTG were from Gold Biotechnology. All other materials
were from Fisher.
Experimental Methods. Protein Expression and Purication. The construction of the plasmid expressing His6-tagged
BaIMPDHL is described in ref 15. The plasmid was
transformed into Escherichia coli BL21 guaB competent
cells.16 His-tagged protein was overexpressed at 18 C for 20
h and puried with Ni-NTA Sepharose beads (GE) at 4 C in
lysis buer [50 mM phosphate buer (pH 8.0), 500 mM KCl, 5
mM imidazole, 1 mM TCEP, and 10% glycerol]. After elution
with 500 mM imidazole, pure protein fractions (>95% as
analyzed by SDSPAGE) were collected and dialyzed rst
against dialysis buer A [50 mM Tris-HCl (pH 8.0), 1 mM
TCEP, 1 mM EDTA, and 300 mM KCl] and then dialyzed
twice against dialysis buer B [50 mM Tris-HCl (pH 8.0), 150
mM KCl, 1 mM DTT, and 3 mM EDTA]. After dialysis, the
protein concentration was determined by the Bradford assay
using IgG as the standard and divided by a factor of 2.6,18 and
the protein was stored at 80 C.
Stopped-Flow Pre-Steady-State Kinetics. Stopped-ow
experiments were performed at 25 C using an Applied
Photophysics SX17MV spectrophotometer. Syringe 1 contained BaIMPDHL protein (nominal concentration of 8 M
subsequently optimized during data analysis) and IMP (2 mM)
in assay buer containing 2.5% (v/v) DMSO. Syringe 2
contained variable concentrations of NAD+ (0.5, 1, 2, 4, 8, 12,
and 16 mM) in assay buer containing 2.5% (v/v) DMSO.
Syringe 2 also optionally contained either 120 M NADH, as a
product inhibitor, or 12 M A110, as an inhibitor of interest.
After preincubation, the contents of both syringes were mixed
in a 1:1 ratio and the reaction progress was monitored by
recording the NADH absorbance at 340 nm. Each co-injection
resulted in 10000 time points spanning from time zero to 2.5 s,
stepping by a t of 0.25 ms. Each individual data trace
(absorbance vs time) for kinetic analysis was obtained as an
average of 12 separate injections. The experiments with NAD+
alone were replicated four times, on separate days, starting from
fresh stock solutions in each daily session. The experiments
with added NADH were replicated twice, as part of sessions 1
and 2. The experiments with added A110 were also replicated
twice, as part of sessions 3 and 4. Thus, the combined stoppedow data set consists of 6720000 raw data points, resulting in
560000 averaged (n = 12) time points, organized into 56
kinetic traces (eight replicated series each containing seven
kinetic traces observed at 0.258 mM NAD+).
Each combined group of kinetic traces, obtained while
simultaneously varying the concentrations of NAD+, NADH,
and A110, was subjected to global regression analysis.19 The
nonlinear least-squares regression model for individual kinetic
traces is dened by eq 1
A = A 0 + rQ ([Q] + [EPQ])
v = v0
[E]0 [I]0 K i* +
([E]0 [I]0 K i*)2 + 4[E]0 K i*

2[E]0
(2)
where square brackets with lower index zero represent total or

analytic concentrations of reactants, v0 is the reaction rate
observed in the absence of inhibitors, and Ki* is the apparent
inhibition constant corresponding to the given kinetic
mechanism (see eqs S25S33, S41, and S42 for denitions
of both v0 and K*i corresponding to the kinetic mechanism in
Scheme 2). Note that at full IMP saturation, [E]0 stands for the
total concentration of the EIMP complex.
For further details regarding data handling and analysis, see
the Supporting Information.
Data Analysis. Nonlinear least-squares regression analysis
was performed by using a custom implementation of the
LevenbergMarquardt algorithm.23,24 For verication, all
regression analyses were repeated by using the hybrid TrustRegion data tting method2527 (algorithm NL2SOL, version
2.3) with the user-supplied Jacobian matrix of rst derivatives.
Initial estimates of microscopic rate constants were discovered
with the aid of the Dierential Evolution global minimization
algorithm.24,28 For further details regarding initial estimates, see
the section 2.2 of the Supporting Information.
(1)
where A is the absorbance (in milli-optical density units, 103

absorbance units) at 340 nm at reaction times of >0.004 s, A0 is
B
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bounds), are fully determined by the available experimental

data. In contrast, a redundant kinetic mechanism contains
rate constants that are assumed to exist on the basis of external
evidence but are not directly supported by experimental data
that are being considered. In this work, we set out to identify
the minimal kinetic mechanism for the available stopped-ow
transient kinetic data and the best-t values, or at least the
lower or upper bounds, for all microscopic rate constants
appearing in the minimal mechanism.
The starting point for the development of a minimal kinetic
model is the redundant mechanism displayed in Scheme 1. The
naming scheme for substrates (A and B) and products (P and
Q) follows IUB/IUPAC recommendations36 as well as
conventions commonly used in classic enzyme kinetic texts.37
The dash in E-P represents the covalent intermediate. This
reaction scheme is based on numerous previously published
reports (for a review, cf. ref 8). The covalent intermediate
species E-P is proposed to exist in two distinct conformations
(open and closed8) which are thought to interconvert
essentially instantaneously on the time scale of the experiment.
By using the trial-and-error approach, eliminating either one
microscopic step at a time or groups of microscopic steps until
all remaining rate constants were fully dened by the data, the
redundant kinetic mechanism in Scheme 1 was reduced to the
minimal kinetic mechanism in Scheme 2. The tilde symbol in
the species name signies that EP could be either the covalent
intermediate on the reaction pathway or the noncovalent
enzymeproduct complex. The stopped-ow transient kinetic
experiment does not provide sucient information to
distinguish between the two scenarios.
Under IMP saturating conditions, the concentration of free
enzyme E is by denition zero, and therefore, the grayed
segment in the top left corner of Scheme 2 is not operational.
Solid black arrows with rate constant names appended to them
represent those microscopic rate constants that can be
unambiguously determined from the stopped-ow transient
kinetic data, not only in terms of their best-t values but also
including the lower and upper bounds. The four dashed arrows
represent those microscopic rate constants (k4, k4, k7, and
k7), for which the lower limit can be determined but not the
upper limit. Rate constants k4 and k4 represent product
inhibition by NADH, whereas rate constants k7 and k7
represent substrate inhibition by NAD+. Both steps can be
characterized as taking place with instantaneous equilibration
(rapid equilibrium approximation). The equilibrium dissociation constants for these two steps are well-dened by the data.
Figure 1 displays 7 of 21 kinetic traces that were all analyzed
as a single global unit.19 The complete set of 21 traces is shown
in Figure S2. In the particular experiment illustrated in Figure 1,
the NAD+ concentration was varied in the presence of added
A110. Each individual experiment involves multiple distinct
phases, as evidenced by the presence of two distinct shoulders
visible in the kinetic trace associated with 8 mM NAD+. The
corresponding instantaneous rate plots (Figure S2, bottom
right panel) further support the observed multiphasic nature of
the transient data. Note that the enzyme and inhibitor
concentrations are comparable in magnitude (tight binding38), which means that simplied pseudo-rst-order
approximation does not hold, and therefore, it would not be
theoretically justied to analyze the transient kinetic data by the
conventional multiexponential analysis. Instead, one must
indeed resort to a global mathematical model formulated as a
system of simultaneous dierential equations.
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.
RESULTS AND DISCUSSION

Stopped-Flow Kinetics. We performed eight replicated
series of stopped-ow experiments in four separate daily
sessions. We used a CBS subdomain-deleted variant of
BaIMPDHL (the same as the crystal structure15). The
enzyme (nominal concentration of 4 M) was preincubated
with xed saturating concentrations of IMP (1 mM; Km = 70
M) and mixed with varied concentrations of NAD+ (0.258
mM) in the presence or absence of NADH (60 M) or A110
(6 M). Representative data are shown in Figure 1 and Figure
S2. Raw data les are available in the Supporting Information.
Minimal Kinetic Mechanism. For the purposes of this
report, a minimal kinetic mechanism is dened as one in
which all individual steps and the associated microscopic rate
constants, or at least their limiting values (the lower or upper
Figure 1. Representative data set from the global t of stopped-ow

transient kinetic data to the kinetic mechanism depicted in Scheme 1.
Concentrations: 4.0 M IMPDH (nominal), 1.0 mM IMP, and 6 M
A110 (nominal). See the labels at the top right for NAD +
concentrations.
C
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Scheme 1. Redundant Kinetic Mechanism of IMPDH
concentration of EAB (blue dashed curve). The two

enzymeinhibitor complexes are formed on dierent time
scales and with dierent abundances at steady state. The
enzymesubstrateinhibitor complex (EAI) is dominant
during the pre-steady-state phase of the experiment up to
approximately 100 ms. The enzymeproductinhibitor complex (EPI) is strongly dominant at steady state (t > 1 s),
although some amount of EAI complex also persists. This
result predicts that in steady-state initial rate measurements
performed under identical experimental conditions (i.e., at
saturating concentrations of IMP), A110 should be identied as
a mixed predominantly uncompetitive inhibitor. On the basis of
the concentration plot in Figure 2, the uncompetitive inhibition
constant can be predicted to be signicantly smaller than the
competitive inhibition constant.
Rate Constant Bounds. To establish the bounds on rate
constants appearing in Scheme 2, the results of nonlinear
regression analysis were averaged from 16 independent
combinatorial replicates (see the Supporting Information for
details regarding combinatorial replication). Table 1 list the
averages (n = 16) and the associated standard deviations from
replicates for the best-t values of rate constants and also for
the corresponding lower and upper bounds evaluated by the 5%
SSQ according to Johnsons empirical method.3234 Similar
results (not shown) were obtained at the more stringent 10%
SSQ condence level.
The microscopic rate constants listed in Table 1 fall into four
categories according to how well they are determined by the
experimental data. In the rst category are three of the four
substrate catalytic constants pointing in the forward direction
(k2, k3, and k5) and also the dissociation rate constants for both
inhibitor binding steps (k8 and k9). All ve rate constants
listed above are very well-dened by the available data, as the
coecient of variation from replicates (n = 16) is <5% in all
cases.
In the second category are the reverse catalytic rate constants
(k2 and k3) and the inhibitor association rate constants (k8
and k9). These four constants are marginally less well
determined, as the corresponding coecient of variation is
approximately between 10 and 25%. However, both the upper
limit and the lower limit of the asymmetric condence interval
are well-dened for all four rate constants.
In the third category are the two microscopic rate constants
that characterize the substrate inhibition step, k7 and k7. Both
best-t values are well-dened, with a CV of <20%. The lower
limit of the condence intervals for k7 and k7 is also very welldened, with a CV of <25%. The upper limit of the condence
Scheme 2. Minimal Kinetic Mechanism of BaIMPDH
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
Figure 2. Evolution of enzyme species concentrations corresponding

to the 1 mM NAD+ kinetic trace displayed in Figure 1. The dominant
enzymeinhibitor complex up to approximately 100 ms is EAI.
However, at steady state (t > 1 s), the dominant enzymeinhibitor
complex is EPI.
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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).
interval is not dened at the 5% SSQ condence level, which

means that the range of plausible values for k7 and k7 spans
from their corresponding lower limits essentially to innity.
However, all pairs of k7 and k7 values that were located within
the 5% SSQ empirical condence intervals maintained a
nearly invariant ratio [k7/k7 = 5.5 mM (Figure S5)]. Thus, the
equilibrium dissociation constant associated with NAD +
substrate inhibition is well-dened by the transient kinetic data.
Finally, in the fourth category are the two rate constants that
characterize product inhibition by NADH, k4 and k4. This is
the only pair of microscopic rate constants for which the best-t
values are poorly dened by the data, as the coecient of
variation is >100% in both cases. However, the ratio of both
rate constants, i.e., the corresponding dissociation equilibrium
constant, remains nearly invariant across all 16 replicated
measurements and is approximately equal to a Kd(EPNH) of 100
M. The upper limits at the 5% SSQ condence level are
undened. Only the lower limits of both k4 and k4 are welldened, with CVs of <25% (Table S4). In particular, the lower
limit of the NADH association rate constant is approximately
k4 > 2 M1 s1. All pairs of k4 and k4 values that were
located within the 5% SSQ empirical condence intervals
maintained a nearly invariant ratio [k4/k4 = 100 M (Figure
S5)]. Thus, the equilibrium dissociation constant associated
with NADH product inhibition is well-dened by the presteady-state kinetic data.
Even though the upper limits of the bimolecular association
rate constants k4 and k7 are not sharply dened by the
available transient kinetic data, those upper limits are imposed
by physical constraints, in particular by diusion control.
Theoretical calculations predict that the diusion-controlled
encounter frequency of an enzyme and a substrate should be
about 109 M1 s1.39 The highest experimentally observed
values39 frequently fall in the range between 106 and 108 M1
s1. The lower limit of k4 (NADH rebinding) is approximately
2 106 M1 s1, which means that NADH rebinding is
extremely rapid. In contrast, the lower limit of k7 (substrate
inhibition by NAD+) is much lower, approximately 4 103 M1
s1.
In summary, of 13 microscopic rate constants that appear in
Scheme 2, 11 rate constants (i.e., all except k4 and k4) were
determined uniquely in terms of their well-reproduced best-t

values. Additionally, nine rate constants have well-dened
upper and lower limits at the 5% SSQ condence level,
according to Johnsons empirical method.33 Johnsons SSQ
method certainly represents a massive improvement over the
conventional and frequently meaningless standard error
method of assessing the uncertainty of nonlinear model
parameters.40 However, it should also be noted that the
empirical SSQ method has no basis in rigorous statistical
theory.29,4143 Instead, the investigator must choose an
arbitrary threshold33 value for SSQ (for example, 5, 10, or
25%), based entirely on subjective personal preferences. Thus,
it is possible that the distinct minima clearly visible in the
likelihood proles44 for k7 and k7 (see Figure S4, bottom
panels) do in fact represent meaningful best-t values.
Importantly, the likelihood proles k4 and k4 (see Figure S4,
top panels) are perfectly at, without even the slightest hint of a
true minimum on the least-squares hypersurface. Nevertheless,
to remain rmly on the safe side, in the analysis of steady-state
initial rates (see below), we have chosen to treat with full
condence only the nine rate constants that have clearly
dened upper and lower limits at 5% SSQ.
It is highly unusual that at least nine microscopic rate
constants should be uniquely determined from a single globally
analyzed transient kinetic data set. In fact, we are not aware of
any published report to that eect, although a large number of
microscopic rate constants have been previously determined
from multiple independent experiments, analyzed separately. In
this fashion, Benkovic et al.45,46 determined 22 microscopic rate
constants in the catalytic mechanisms of dihydrofolate
reductase. Similarly, Anderson et al.47 determined 12 microscopic rate constants in eight separate rapid quench kinetic
experiments (see Figures 18 in ref 47), each of which was
focused on a particular subset of the overall 12-step mechanism.
The largest number of microscopic rate constants ever
determined in a global t of a single global data set is six, as
reported by Schroeder et al.48,49 The challenges of successful
model identication grow very much faster than linearly with an
increase in the number of adjustable model parameters. Thus, a
kinetic mechanism containing either 9 or even 11 adjustable
rate constants is signicantly more dicult to establish by
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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.
and 1000 M1 s1 (kon). The purpose was to determine the

extent (if any) to which the best-t values of the remaining rate
constants appearing in Scheme 2 are aected by the arbitrary
choice of kon = k4 = k7. The results are summarized in Table
S5.
Within the examined range of kon values, the best-t values of
the corresponding koff rate constants were such that the Kd =
koff/kon remained invariant. Specically for product inhibition
by NADH, the dissociation equilibrium constant k4/k4
remained within 5% of 100 M. Similarly for substrate
inhibition by NAD+, the dissociation equilibrium constant
k7/k7 remained within 10% of 5.5 mM. Importantly, the
remaining microscopic rate constants appearing in Scheme 2
varied by <5% in response to arbitrary variations in the
assumed kon value spanning 3 orders of magnitude. Thus, the
ultimate values of microscopic rate constants that were
subjected to subsequent validation by steady-state initial rate
kinetic measurements are those that are listed in the right-most
column of Table S5. These values assume that the rapid
equilibrium assumption is suciently well characterized by a kon
of 100 M1 s1.
tting a single data set, compared to a kinetic mechanism with

six microscopic rate constants, the largest number reported so
far.48,49 Therefore, to stress-test our regression model in terms
of reproducibility, we have performed 16 replicated regression
analyses, by utilizing the combinatorial mix-and-match
method described above.
The successful determination of at least nine microscopic
rate constants in the global analysis19 of a single combined
transient kinetic data set was almost certainly facilitated by the
fact that the UV/vis spectrophotometric signal was sensitive
not only to the presence of the nal reaction product (NADH,
Q in Scheme 2) but also very importantly to the presence of
the enzymeproduct complex (E-PQ in Scheme 2; see also eq
1).
Partially Constrained Model. On the basis of the fact that
only lower limits can be determined for kinetic constants that
characterize either substrate inhibition by NADH (k4 and k4)
or product inhibition by NAD+ (k7 and k7), the kinetic model
shown in Scheme 2 was stress-tested in a series of regression
analyses in which association rate constants k4 and k7 were
both assigned arbitrarily chosen rapid equilibrium values. The
ve arbitrarily chosen rate constant values were 10, 20, 50, 100,
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the observed reaction rate (approximately 0.4 10 3

dimensionless absorbance unit per second) corresponds to
the chemical reaction rate of approximately 0.4/6.22 = 0.064
M/s in NADH formation.
The results displayed graphically in Figure 3 show that the 13
microscopic rate constants determined by the stopped-ow
transient kinetic measurements predict the steady-state initial
rate data reasonably well. For example, the position of the
maximum on the NAD+ saturation curve (at approximately 1.5
mM NAD+), in the top left panel, is well predicted, as is the
slope of the downward portion due to substrate inhibition. In
the top right panel of Figure 3, there is good agreement
between the predicted (curves) and observed (symbols)
product inhibition eect of NADH. The bottom left panel
shows the predicted versus observed inhibition eect of A110
at relatively low concentrations of NAD+. Again, the relative
spacing of the substrate saturation curves is described well by
the theoretical model derived from the stopped-ow data. The
same applies to the bottom right panel of Figure 3, displaying
the inhibitory eect of A110 at relatively high concentrations of
NAD+.
Predicted versus Observed Composite Kinetic Constant.
On the basis of the best-t values of microscopic rate constants
determined in the stopped-transient kinetic experiments (see
Table 1) and given the denition of steady-state kinetic
constants as shown in eqs S25S33, the predicted values of
kinetic constants are listed in Table 2 (predicted column). To
Steady-State Initial Rate Kinetics. Predicted versus

Observed Reaction Rates. Steady-state initial rates were
determined in four types of experiments, depending on the
varied reaction component(s). In all these experiments, the
substrate concentration was held xed at a saturating IMP
concentration of 1.0 mM. The fact that 1.0 mM IMP is indeed
saturating can be established by inspection of Figure S9 (top
left panel). Note that two IMP saturation curves observed at
0.75 and 1.0 mM IMP are virtually indistinguishable.
In the rst type of experiment, aimed at establishing the
cofactor saturation curve, the NAD+ concentration was varied
between 0.1 and 8.0 mM, in the absence of either NADH as the
product inhibitor or A110 as the inhibitor of interest. This
experiment was performed in triplicate. In the second type of
experiment, aimed at the product inhibition eect of NADH,
the NAD+ concentration was varied between 0.1 and 1.0 mM at
various levels of NADH (0, 50, 100, and 150 M). In the third
type of experiment, aimed at the inhibition properties of A110,
the NAD+ concentration was varied between 0.12 and 1.2 mM
at various levels of A110 (0, 15, 30, 60, 120, and 180 nM). The
fourth and nal type of experiment was a variation on the
immediately preceding experiment type and involved relatively
high concentrations of NAD+, varied between 1.0 and 8.0 mM
at various levels of A110 (0, 15, 30, 60, 90, and 150 nM).
The experimental data from all four types of experiment were
combined into a single superset of data and analyzed by the
global-t19 method. The nonlinear regression model was eq 2.
The various algebraic terms that dene the uninhibited rate v0,
eq S41, and the apparent inhibition constant, Ki*, eq S42, are
dened as shown in eqs S25S33. In eqs S25S33, the
assumed values of all microscopic rate constants were those
obtained in the stopped-ow transient kinetic study: k2 =
0.0318 M1 s1; k2 = 10.9 s1; k3 = 82.1 s1; k3 = 44.3 s1; k4
= 11500 s1; k4 = 100 M1 s1 (rapid equilibrium
approximation); k5 = 13.6 s1; k7 = 100 M1 s1 (rapid
equilibrium approximation); k7 = 566000 s1; k8 = 13.9 M1
s1; k8 = 11.0 s1; k9 = 5.51 M1 s1; and k9 = 0.27 s1.
Importantly, in this rst round of initial rate analysis, all
microscopic rate constants listed above were held xed at values
derived from the stopped-ow experiment. The only adjustable
model parameter in the regression equation (eq 2) was the
active enzyme concentration, [E]0. The enzyme concentration
was optimized locally for each particular type of experiment,
because the dierent steady-state experiments were performed
over a period of approximately one year, and thus, the active
enzyme concentration might have changed slightly over time.
The nominal enzyme concentration was 14 nM, and the best-t
values of the active site concentration varied from 8.4 to 10.7
nM.
Given the fact that all rate constants were held xed in the
regression and only the enzyme concentration was optimized,
the highly restricted t of the combined initial rate data is
merely a comparison between the observed initial rates and
those that are predicted by the theoretical model derived form
the stopped-ow experiment. The results are shown in Figure 3.
The full text of the requisite DynaFit input le is listed in
Appendix A.2.2; the best-t values of [E]0 are listed in Table S6.
Note that that throughout this report the reaction rates are
expressed in directly observable absorbance units per second,
rather than in concentration units. The main reason for this
choice is that, in particular in the analysis of stopped-ow data,
the molar response coecients are generally treated as
adjustable model parameters. In the specic case of Figure 3,
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.
validate this prediction, the combined initial rate data were

again t globally19 to eq 2, this time while treating all kinetic
constants [kcat, Km(B), Ki(I), Ki(I,B), Ki(B), Ki(Q), and Ki(Q,B)] as
adjustable model parameters.
The best-t values obtained in this second round of tting
the initial rate data are listed in Table 2 and are shown
graphically in Figure S9. The results of the t are in good
agreement with the values predicted from the theoretical model
derived from the stopped-ow transient kinetic experiment.
The uncompetitive inhibition constant Ki(I,B) predicted
from stopped-ow measurements is 57 nM; the experimentally
observed value from initial rate measurements was 51 2 nM,
with the 95% condence level interval spanning 4754 nM. In
previous reports of the kinetics of full length BaIMPDH, the
uncompetitive inhibition constant for A110 with respect to
G
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].
variable NAD+ concentrations was reported to be 58 4 nM13

and 57 7 nM.50 The IC50 of A110 inhibiting BaIMPDHL
was reported to be 43 3 nM at 1.0 mM IMP and 1.5 mM
NAD+.
The competitive inhibition constant Ki(I) predicted from
stopped-ow measurements is 0.8 M; the experimentally
observed value from initial rate measurements is 0.5 0.2 M,
with the 95% condence level interval spanning 0.31.5 M.
Thus, the initial rate experiments conrm the prediction of
inhibition mode based on the results of the stopped-ow
experiments. A110 was predicted to behave as a mixed
predominantly uncompetitive inhibitor of BaIMPDHL, and
this is in fact conrmed by initial rate measurements.
Predicted versus Observed Kinetic Isotope Eect. The
stopped-ow kinetic results yielded a distinctly non-zero best-t
value of the EAB EA + B dissociation rate constant, k2
(see Table 1). However, k2 appears to be eectively zero (i.e.,
NAD+ is a sticky substrate) for at least some IMPDHs.51 To
verify the stopped-ow kinetic result in this respect, the D(kcat/
Km) isotope eect was determined by assaying BaIMPDHL
with saturating [1H]IMP and [2D]IMP concentrations. The
results are reported in detail in section 3.3.4 of the Supporting
Information (Table S7).
Briey, the value of k2 listed in Table 1 predicts that D(kcat/
Km) should be 1. On the other hand, if k2 were negligibly
small (sticky substrate), then the predicted value of D(kcat/
Km) should be by denition equal to 1. The observed value is
2.4 0.1. The isotope eect on kcat/Km(B) conrms that that
NAD+ dissociates readily from the EAB complex, in
agreement with the pre-steady-state kinetic results.
A110 plus XMP Double-Inhibitor Experiment. To determine if A110 binds to E-P or EP, the apparent inhibition
constant for A110 was determined at varying concentrations of
XMP (0.1252.0 mM) in the presence of subsaturating
concentrations of IMP (0.2 mM) and NAD+ (1.5 mM).
Experimental doseresponse curves were tted to eq 2, where
[E]0 was held xed at 10 nM while v0 and Ki* were treated as
optimized model parameters. The raw experimental data are
shown in Figure 4A. The best-t values of the apparent
inhibition constant at various XMP concentrations and the
associated formal standard errors from nonlinear regression are

listed in Table S8. The apparent inhibition constant increased
approximately linearly with an increase in product concentration (Figure 4B). This observation suggests that EXMP
does not form a high-anity complex with A110. The observed
changes in K*i with [P] were subjected to weighted linear
regression analysis. The results of a linear t are shown
graphically in Figure 4B. The dimensionless slope of the linear
regression line was (7.0 0.6) 105; the intercept
representing K*i at [P] = 0 was 137 6 nM.
Figure S14 shows the result of a heuristic simulation
designed to allow an interpretation of the experimentally
observed dependence. The simulation results can be
summarized as follows. If the binding anity of A110 for the
noncovalent intermediate EP were greater than the binding
anity for the covalent complex E-P, the plot of K*i versus [P]
would be sloping downward. If both binding anities were
nearly identical, the plot would be approximately horizontal.
Finally, if the binding anity of A110 toward the covalent
intermediate E-P were dominant, the plot of K*i versus [P]
would be sloping upward. The experimentally observed plot has
an upward slope, signifying that A110 does not bind
predominantly to the noncovalent product complex but instead
binds preferentially to the covalent intermediate. The
mathematical details are explained in section 3.3.5 of the
Supporting Information. A very approximate estimate of the
ratio of the two relevant inhibition constants suggests that the
binding to EP might be at least an order of magnitude weaker
than the binding to E-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
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.
similar to that found in IMPDHs from from many pathogenic

bacteria, including B. anthracis, Campylobacter jejuni, Cl.
perf ringes, Streptococus pyogenes, Heliobacter pylori, and M.
tuberculosis.5 Despite this similarity, the anity of the inhibitors
for these enzymes can vary by 1001000-fold. Structures of
several inhibitors bound to EIMP complexes have been
determined, but these structures do not reveal the basis for this
surprising variation. Here we have delineated the kinetic
mechanism of one representative inhibitor, A110, as shown in
Scheme 3, which displays the dominant inhibitor binding mode.
According to Scheme 3, this compound binds preferentially

to the covalent intermediate. Thus, the crystal structures do not
represent the high-anity enzymeinhibitor complex, which
may explain why they do not provide insight into the varied
spectrum of IMPDH inhibitors.
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.biochem.6b00265.
Description of mathematical and statistical procedures
(PDF)
Raw experimental data (CSV text format) and DynaFit
input scripts (ASCII text format) (ZIP)
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Scheme 3
Article
AUTHOR INFORMATION
Corresponding Author
*E-mail: hedstrom@brandeis.edu. Phone: +1 (781) 736-2333.

Fax: +1 (781) 736-2349.
Present Addresses
Y.W.: Sanford-Burnham-Prebys Medical Discovery Institute,

La Jolla, CA 92037.
G.M.: Department of Pharmaceutics, Indian Institute of

Technology, Banaras Hindu University, Varanasi 221005, India.
Funding
This work was supported by National Institutes of Health

Grants AI093459 and GM054403 (to L.H.).
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
The authors thank Cynthia Tung for assistance with the
synthesis of A110.
I
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J
Inhibition of IMPDH from Bacillus anthracis. Mechanism

revealed by pre-steady state kinetics
S UPPLEMENTARY I NFORMATION
Yang Weia,1 , Petr Kuzmicb , Runhan Yua , Gyan Modia , Lizbeth Hedstrom,a
a Departments
of Biology and Chemistry, Brandeis University, Waltham, Massachusetts

b BioKin Ltd., Watertown, Massachusetts
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
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3
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3 Steady-state initial rate kinetics

16
3.1 Experimental data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.2 Theoretical model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Corresponding
1 Current
author
address: Sanford-Burnham Institute, La Jolla, California
Biochemistry Ms. No bi-2016-00265u.R2
Supporting Information
3.3
3.4
3.2.1 Classical rate equation (King-Altman & Cleland)

3.2.2 Tight binding rate equation (Morrison) . . . . .
Steady-state initial rate results . . . . . . . . . . . . . . .
3.3.1 Predicted vs. observed initial rates . . . . . . . . .
3.3.2 Predicted vs. observed kinetic constants . . . . . .
3.3.3 Diagnostic Eadie-Hofstee plots . . . . . . . . . . .
3.3.4 Predicted vs. observed H/D isotope effect . . . . .
3.3.5 Double inhibition experiment . . . . . . . . . . .
Steady-state kinetics: Summary and conclusions . . . . . .
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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 . . . . . . .
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44
44
46
46
47
49
50
51
52
52
53
54
B Raw data files and DynaFit script files

B.1 Installation instructions . . . . . . .
B.2 Organization of files and directories
B.2.1 DynaFit script files . . . . .
B.2.2 Raw experimental data files
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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
new batch of NAD+
Table S1: Organization of replicated stopped-flow experiments. Replicate labels correspond to

available raw data file names. For details see text.
2 Abbreviations: DE, Differential Evolution algorithm; IMP, inosine 5-monophosphate; IMPDH, IMP dehydrogenase; BaIMPDH, IMPDH from Bacillus anthracis; BaIMPDHL, BaIMPDH Glu92-Arg220 deletion mutant; MPA,
mycophenolic acid; ODE, ordinary differential equations; SSQ, residual sum of squares; SSQ, relative change in SSQ
above the minimum value; XMP, xanthosine 5-monophosphate
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
Table S2: Combinatorial replication scheme. Each of N, H, and I replicates consists of

seven kinetic traces obtained at [NAD+ ] = 0.25, 0.5, 1, 2, 4, 6, 8 mM, either in the absence of
any other component (N) or in the presence of [NADH] = 60 M (H), or in the presence of
[A110] = 6 M (I). For details see text.
All microscopic rate constants reported in this document, as well as the lower and upper limits
of their respective confidence intervals, are simple averages and standard deviations determined
from 16 (combinatorially) replicated global data sets as shown in Table S2.
2.2. Theoretical model
The goal of our model-building effort was to identify a minimal mechanistic model for the
available transient kinetic data. A minimal kinetic model is a reaction scheme where all microscopic rate constants (or at least their well defined limiting values) can be both structurally
and practically identified [4] from the available experimental data. A hybrid modeling strategy
combined (a) heuristic variations on previously published kinetic models [5, 6] and (b) global
least-squares minimizations using the Differential Evolution (DE) algorithm [7]. For details regarding DE see [2, pp. 264267]. The total evolutionary computing time was approximately 300
4
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.
Regression equation and model parameters

The nonlinear least-squares regression model for individual kinetic traces is defined by Eqn
(S1), where A is the absorbance (in mOD, 103 dimensionless absorbance units) at 340 nm at
reaction time t > 0.004 sec; A0 is the adjustable baseline offset at t0 = 0.004 sec, essentially a
property of the instrument; rQ = 6.22 mOD/M is the molar response coefficient of NADH3 ; [Q]
is the concentration of NADH at time t; and [EPQ] is the concentration of the ternary complex
E-XMP NADH.
A = A0 + rQ ([Q] + [EPQ])
(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
k2 [EA][B] + k2 [EAB] + k5 [EP] k8 [EA][I] + k8 [EAI]
(S2)
d[B]
dt
k2 [EA][B] + k2 [EAB] k7 [EP][B] + k7 [EPB]
(S3)
d[EAB]
dt
+k2 [EA][B] k2 [EAB] k3 [EAB] + k3 [EPQ]
(S4)
d[EPQ]
dt
+k3 [EAB] k3 [EPQ] k4 [EPQ] + k4 [EP][Q]
(S5)
d[EP]
dt
+k4 [EPQ] k4 [EP][Q] k5 [EP] k7 [EP][B] + k7 [EPB]

k9 [EP][I] + k9 [EPI]
(S6)
d[Q]
dt
+k4 [EPQ] k4 [EP][Q]
(S7)
d[P]
dt
+k5 [EP]
(S8)
d[EPB]
dt
+k7 [EP][B] k7 [EPB]
(S9)
d[I]
dt
k8 [EA][I] + k8 [EAI] k9 [EP][I] + k9 [EPI]
(S10)
d[EAI]
dt
+k8 [EA][I] k8 [EAI]
(S11)
d[EPI]
dt
+k9 [EP][I] k9 [EPI]
(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
applies to trace no.
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.
Initial estimates of model parameters

In general, the nonlinear least-squares regression procedure requires that the investigator
provides initial estimates of adjustable model parameters [8]. In unfavorable cases, the initial
estimates must be surprisingly close the true values (which of course are not known at the outset),
otherwise the least-squares minimization algorithm ends up in a false minimum. In preliminary
data analyses (results not shown), the fitting model was found exquisitely vulnerable to falling
into numerous false minima on the least-squares hypersurface. False convergence into a local
least-squares minimum was reached frequently even when certain rate constants were shifted
from their optimal value by less than a factor of 10. This local minimum problem was overcome
by deploying the DE algorithm as a global least-squares minimizer [7]. The final set of nearly
optimal initial estimates is specified in the DynaFit script listed in Appendix A.1:
[constants]
k2 = 0.01 ??
k3 = 100 ??
k4 = 1000 ??
k5 = 10 ??
k7 = 0.01 ??
k9 = 10 ??
k9 = 1 ??
,
,
,
k-2 = 10 ??
k-3 = 10 ??
k-4 = 10 ??
,
,
,
k-7 = 100 ??
k-8 = 10 ??
k-9 = 0.1 ??
Empirical confidence intervals

The double question mark in the code fragment listed above signifies that the given rate constant are to be subjected to a systematic search to determine the asymmetric confidence interval
7
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
empirical interval, 5 % SSQ increase

1.1
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:
the lower limits for rate constant k4 and k4 ;

the lower limits for rate constant k7 and k7 ;
the equilibrium constant Kd(EP.NH) = k4 / k4 = 100 M; and
the equilibrium constant Kd(EP.N) = k7 / k7 = 5.5 mM.
2.3.2. Combinatorial replicates

To our knowledge it is highly unusual that more than at least nine microscopic rate constants
should be uniquely determined from a single pre-steady state kinetic data set. In fact, we are not
aware of any published report to that effect. Therefore, in order to stress-test our regression model
in terms of reproducibility, we have performed 16 replicated regression analyses, by utilizing the
combinatorial mix-and-match method described above. It should be noted that all series of
712 = 84 stopped-flow runs (7 varied NAD+ concentrations, 12 replicated injections to generate
a single curve) were performed on different days, starting from fresh stock solutions. The bestfit values of all 13 adjustable rate constants are summarized in Figure S6. The numbering of
combinatorial replicates on the horizontal axis corresponds to the rows of Table S2.
The upper left panel in Figure S6 groups together all microscopic rate constants that appear in
the substrate branch of the kinetic scheme in Figure S1, except for the NADH product inhibition
11
1.1

search
bounds
best-fit
1.04
SSQrel
1.02
1.05
SSQrel
1.06
1.08
search
bounds
best-fit
100
1000 10000 1000001e+0061e+0071e+008
10
100
1000 10000 1000001e+006
k4
k-4
1.05
SSQrel
1.15
search
bounds
best-fit
1.1
1.15
1.1
1.05
SSQrel

search
bounds
best-fit
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
k-4, M-1 s-1
10000
search k3
search k-3
100
1000
10000
100000
1e+006
0.01
k4, s-1
0.1
k7, M-1 s-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
NAD product inhibition
k7
k-7
k7/k-7
log10 (rate constant)
k2
k-2
k3
k-3
k5
-2
-1
catalytic cycle except NADH product inhibition
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.
Figure S6: Reproducibility of rate constant determination in 16 combinatorial replicates. For

details see text.
The particular fixed values were kon = k4 = k7 = 10, 20, 50, 100, 1000 M1 s1 , i.e., spanning from 107 to 109 M1 s1 . In either of the five cases, the best-fit model curves overlaid on
the experimental data as well as the corresponding residual plots were visually indistinguishable
from Figure S2 (results not shown). As expected from the confidence interval profile shown in
Figure S4, the best-fit residual sum of squares was almost exactly 5% higher compare to the
first round of analysis where both k7 and k7 were treated as adjustable model parameters. The
numerical results are summarized in Table S5. The results for kon = 1000 M1 s1 were exactly
identical to those obtained for kon = 100 M1 s1 and are not shown.
The results summarized in Table S5 indicate that the average (n = 16) best-fit values of microscopic rate constants appearing Figure S1 are largely insensitive to the assumed, fixed values
of the association rate constants k4 and k7 . The relative changes going from kon = 10 M1 s1
to kon = 100 M1 s1 amount to at most a few percentage points. The same applies to the various
14
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
kon > 106 M-1s-1
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.
NAD+ varied alone, in the absence of any additional component.

NAD+ varied in the presence of various amounts of NADH (product inhibition).
NAD+ varied in the presence of various amounts of A110.
NAD+ varied in the presence of either 1 H- or 2 H-IMP.
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
an extended period of time showed some non-negligible variations, approximately 10%20%.

Therefore the actual enzyme concentration in the global fit of initial rate data was treated as an
adjustable model parameter.
Finally, one additional initial rate data set (the double-inhibition experiment) was collected
at sub-saturating concentration of the IMP substrate ([S]0 = 0.2 mM) and therefore it is not fully
compatible with the transient kinetic experiments. In that special case, both A110 and XMP were
both varied simultaneously as inhibitors.
3.2. Theoretical model
3.2.1. Classical rate equation (King-Altman & Cleland)
In order to facilitate the derivation of the classical initial rate equation (i.e., one in which
possible inhibitor depletion due to tight binding conditions is ignored), the DynaFit software
[2] was presented with mechanism specification listed below:
[task]
...
data = rates
approximation = king-altman
[mechanism]
enzyme E.A
reaction B ---> P + Q
modifiers I
E.A + B <==> E.A.B
:
k2
E.A.B <==> E.P.Q
:
k3
E.P.Q <==> E.P + Q
:
k4
E.P ---> E.A + P
:
k5
E.P + B <==> E.P.B
:
k7
E.A + I <==> E.A.I
:
k8
E.P + I <==> E.P.I
:
k9
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
...
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)
D = d1 + d2 [I] + d3 [Q] + d4 [B] + d5 [Q][I] + d6 [B][I] + d7 [B][Q]

+d8 [B]2
(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
See Eqn. (12) in [17, p. 272].
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)
3.3. Steady-state initial rate results

3.3.1. Predicted vs. observed initial rates
The fitting model for transient kinetic data contains 11 adjustable microscopic rate constants
(Table S5) in addition to the fixed values of k4 and k7 . The question arises whether or not the
kinetic model with such large degree of flexibility can accurately predict steady state initial rates
determined in a series of independent experiments under identical conditions. The DynaFit script
listed in Appendix A.2.2 was designed to answer that question.
22
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
; <== insert this input line
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
NAD (low) + A110
1000
NAD (high) + A110
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
NAD (Eadie-Hofstee plot)
NAD + NADH (Eadie-Hofstee plot)
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+]
NAD (low) + A110 (Eadie-Hofstee plot)
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+]
NAD (high) + A110 (Eadie-Hofstee plot)

I = 0 nM
I = 15
I = 30
I = 60
I = 120
I = 180
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:
The enzyme concentration was [E] = 10 nM.

The IMP substrate concentration was [A] = 0.2 mM.
The NAD+ cofactor concentration was [B] = 1.5 mM.
The A110 inhibitor was varied as [I] = 0, 15, 30, 60, 120, 180 nM
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
XMP as product inhibitor was varied as [P] = 0, 0.125, 0.25, 0.5, 1, 2 mM

At each given [XMP] concentration, the apparent inhibition constant for A110, Ki , was
determined by a fit of [I] vs. initial rate data to the Morrison Eqn (S40).
A plot of Ki vs. [XMP] was constructed to ascertain whether Ki changes with [XMP] and,
if it does, in what fashion.
The basic premise of the double-inhibition experiment is that if Ki for A110 were to show
a detectable decrease (stronger binding) with increasing [XMP], it would be reasonable to conclude that A110 binds preferentially to the noncovalent complex E.P. The reasoning is that at
elevated levels of [XMP], more E.P is available for A110 binding and therefore the inhibition
potency would appear enhanced. In contrast, if Ki for A110 were to show a detectable increase
(weaker binding) with increasing [XMP], it would be reasonable to conclude that A110 binds
preferentially to the covalent intermediate E-P.
In order to allow for XMP product inhibition to be observable experimentally, the IMP substrate concentration was decreased from the saturating concentration [A] = 1.0 mM, used every(app)
where else in this report, to [A] = 200 M. The apparent Michaelis constant Km(A) at [B] = 1.5
mM is approximately 60 M (see Appendix A.2.4 and Figure S12A). Therefore the [A] = 200 M
is clearly sub-saturating. A back-of-the-envelope calculation shows that under these conditions
approximately 23% of the enzyme is the free, unliganded state. This allows XMP to express
its product-inhibition potential. The apparent inhibition constant of XMP (P) at [A] = 200 M
and [B] = 1.5 mM is approximately 1.1 mM (see Appendix A.2.5 and Figure S12B). This allows
XMP to affect easily detectable product inhibition within the concentration range chosen for the
double-inhibitor experiment, [P]max = 2.0 mM.
A
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
IMP : Km(app) = (61 +/- 3) M
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
Experimental determination of Ki vs. [P]

Inhibitor dose-response curves observed at various fixed concentrations of [P] and [A] =
200 M, [B] = 1.5 mM were fit to the Morrison Eqn (S40) to determine the apparent inhibition
constant, Ki . The requisite DynaFit script is listed in Appendix A.2.6. The results of fit are
illustrated graphically in Figure 4A of the main manuscript. The best-fit values of the apparent
inhibition constants vs. product concentrations are listed in Table S8.
[XMP], M
0
125
250
500
1000
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
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
Figure S13: Assumed kinetic mechanism for the double-inhibitor experiment.

[mechanism]
reaction S + N ---> P + NH
modifiers I
; Catalysis:
E + S <==> E.S
:
E.S + N <==> E.S.N
:
E.S.N <==> E-P.NH
:
E-P.NH <==> E-P + NH
:
E-P ---> E.P
:
E.P <==> E + P
:
E-P + N <==> E-P.N
:
; Inhibition:
E.S + I <==> E.S.I
:
E-P + I <==> E-P.I
:
E.P + I <==> E.P.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
The DynaFit software derived the following algebraic model:

N
D
v =
[E]0
n1 [A][B]
D =
(S55)
d1 + d2 [P] + d3 [Q] + d4 [B] + d5 [A]
(S56)
+d6 [P][I] + d7 [Q][P] + d8 [B][P] + d9 [A][I] + d10 [A][Q]

+d11 [A][B] + d12 [Q][P][I] + d13 [B][P][I] + d14 [A][Q][I]
+d15 [A][B][I] + d16 [A][B][Q] + d17 [A][B]2
32
(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
k1 k2 k3 k4 (k6 k9 k10 + k5 k9 k10 )

k1 k5 k6 k9 k10 (k2 k3 + k2 k4 + k3 k4 )
33
(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)
Derived kinetic constants
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 )
(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
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) =
Rate equation cast in composite kinetic constants

The following algebraic model was derived by algebraic rearrangements of the computergenerated model listed above:
N
D
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)
Derivation of the apparent inhibition constant

Applying the method of Morrison [17], in the general case when NADH could act as product
inhibitor ([Q] > 0), the apparent inhibition constant for I is obtained as shown in Eqn (S94).
Ki
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
Linear fit of simulated Ki

Ki(I,P) = 5.12
Ki(I,P) = 2.56
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
depending on the assumed value of Ki(I,P) .

If inhibitor binding to the covalent intermediate E-P were stronger than the binding to the
noncovalent product complex E.P, the apparent inhibition Ki would increase with [P]. The
trend changes from approximately hyperbolic to linear.
If the binding to the covalent intermediate E-P were weaker than the binding to the noncovalent product complex E.P, the apparent inhibition Ki would decrease with [P]. The
trend would be approximately hyperbolic.
In the range approximately between Ki(I,P) = 80 nM and Ki(I,P) = 160 nM, the plot of Ki vs.
[P] would appear approximately horizontal.
Linear fit of simulated Ki values
The plots of simulated Ki vs. [P] displayed in Figure S14A are clearly nonlinear. However,
in an actual experiment the nonlinearity might be somewhat obscured by inevitable experimental error. Once random error is introduced it seems reasonable to assume that experimentally
observed plots of Ki vs. [P] might appear essentially linear. It is therefore of interest to perform linear fit of at least some relevant simulated values displayed in Figure S14A. The requisite
DynaFit script is listed in Appendix A.2.8.
The results of fit are shown in Figure S14B. Recall that the assumed value of the inhibition
constant Ki(I,A,B) , which measures the binding affinity of A110 toward the covalent intermediate
E-P is 60 nM. Figure S14B shows the various outcomes simulated at values of the inhibition
constant Ki(I,P) , which measures the binding affinity of A110 toward the noncovalent complex
E.P. The most salient features of the linear regression analyses match the the pattern described
above, in the sense that the slope of the regression line depends on the relative binding affinities
of I toward the covalent intermediate vs. the noncovalent complex. Most importantly if I binds
preferentially to the covalent intermediate, the slope of the regression line is positive.
Comparison with experiment
Experimentally observed values of the apparent inhibition constant for A110 under relevant
experimental conditions are listed in Table S8 above. Those values were subjected to linear
regression analysis to determine the experimental slope of the plot of Ki vs. [P]. The requisite
DynaFit script is listed in Appendix A.2.9. The script performs a weighted regression using the
linear model,
Ki = Ki(0) + A [XMP]
where Ki(0) is the value of Ki at zero [XMP] concentration and and A is the slope. The
weighting scheme uses the obviously unequal experimental errors associated with each particular
measurement of Ki at at different values of [XMP]. The results of fit are shown graphically in
Figure 4A (main manuscript).
The best fit values are are Ki = (0.137 0.006) M and A = (7.0 0.6) 105 (a dimensionless value). A comparison between the experimentally observed slope with slopes simulated at
various assumed values of the inhibition constant Ki(I,P) is shown in Table S9. The experimentally
observed slope is inserted between two immediately adjacent slope values.
The results summarized in Table S9 would seem to lead to the tentative conclusion that that
the most probable value of Ki(I,P) , which measures the binding affinity of A110 toward the noncovalent complex E.P, lies in the vicinity of 0.5 M (between 0.32 and 0.64 M). This value is
38
Ki(I,P) , M
5.12
2.56
1.28
0.64
experiment
0.32
0.16
slope A 105 Std. Error

11.3
10.8
9.8
8.2
7.0
5.5
1.9
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 |
44
label
label
label
label
0.25
0.5
1
2
shift 12 | column 6 | offset auto ? | conc B = 4000 | label 4

shift 20 | column 7 | offset auto ? | conc B = 6000 | label 6
shift 28 | column 8 | offset auto ? | conc B = 8000 | label 8 mM
;
; Product inhibition by NADH (60 uM):
;
graph [NADH] = 60 {/Symbol m}M, [A110] = 0
maximum
0.5
sheet
H1.csv
shift 0 | column 2 | offset -373 ? | conc Q = 60, B = 250 | label 0.25
shift 1 | column 3 | offset -372 ? | conc Q = 60, B = 500 | label 0.5
shift 2 | column 4 | offset -371 ? | conc Q = 60, B = 1000 | label 1
shift 28 | column 8 | offset -345 ? | conc Q = 60, B = 8000 | label 8 mM
;
; Inhibition by A110 (6 uM):
;
graph [NADH] = 0, [A110] = 6 {/Symbol m}M
maximum
off
sheet
I1.csv
shift 0 | column 2 | offset auto ? | conc I = 1 * In, B = 250 | label 0.25
shift 1 | column 3 | offset auto ? | conc I = 1 * In, B = 500 | label 0.5
shift 2 | column 4 | offset auto ? | conc I = 1 * In, B = 1000 | label 1
shift 12 | column 8 | offset auto ? | conc I = 1 * In, B = 8000 | label 8 mM
[output]
directory ./project/impdh/01/progress/output/fit-N1H1I1
[settings]
{Filter}
ZeroBaselineSignal = y
XMin = 0.004
TimeFirstMesh = 0.003
PointsPerDataset = 25
ExponentialSpacing = y
{Output}
WriteTeX = y
WriteEPS = y
XAxisLabel = time, s
YAxisLabel = {/Symbol D}A_{340}, mOD
SquaresIncreasePercent = 5
[end]
45
A.2. Global fit of initial rates

A.2.1. Predict kinetic constants: Classical rate equation
This DynaFit [2] script performs global fit of combined initial rate data to the classical rate
equation automatically derived by the King-Altman method. All microscopic rate constants are
held fixed at values determined by the analysis of stopped-flow data. Only the enzyme active site
concentration is optimized. Any tight binding is ignored.
Compare observed initial rates with those predicted from the
minimal transient kinetic model based on stopped-flow data.
King-Altman approximation: any "tight binding" is ignored.
;______________________________________________________________________
[task]
task = fit
data = rates
[mechanism]
enzyme E.A
modifiers I
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 = 0.0318 ,
k-2 = 10.9
k3 = 82.1
,
k-3 = 44.3
k4 = 11500
,
k-4 = 100
k5 = 13.6
k7 = 100
,
k-7 = 566000
k8 = 13.9
,
k-8 = 11
k9 = 5.51
,
k-9 = 0.27
[responses]
Q = 6.22
[parameters]
cE1 = 0.01 ?
cE2 = 0.01 ?
cE3 = 0.01 ?
cE4 = 0.01 ?
cE5 = 0.01 ?
[data]
variable B
directory ./project/IMPDH/01/rates/data
graph NAD
sheet nad-b.csv
column 2 | conc E.A = 1 * cE1 | label A = 750 uM
column 3 | conc E.A = 1 * cE1 | label A = 1000 (a)
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 =
graph NAD (low) + A110
sheet nad-a110-d.csv
column 2 | conc E.A = 1 * cE4, I = 0.000 | label I
graph NAD (high) + A110
sheet nad-a110-a.csv
[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
A.2.2. Predict kinetic constants: Tight binding rate equation

This DynaFit [2] script performs global fit of combined initial rate data to the Morrison
equation defined in Eqns (S40)(S42). All kinetic constants are held fixed at values determined
by the analysis of stopped-flow data. Only the enzyme active site concentration is optimized.
The model is formulated in terms of composite kinetic constants. Tight binding is properly
accounted for.
Compare observed initial rates with those predicted from the
minimal transient kinetic model based on stopped-flow data.
;______________________________________________________________________
[task]
task = fit
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
column 2 | param E = 1 * cE2 | label A = 1.0 (c)

graph NAD + NADH
sheet nad-nadh.csv
column 2 | param E = 1 * cE3, Q =
0 | label Q = 0 mM
column 3 | param E = 1 * cE3, Q = 50 | label Q = 0.05
graph NAD (low) + A110
sheet nad-a110-d.csv
column 2 | param E = 1 * cE4, I = 0.000 | label I = 0 nM
column 3 | param E = 1 * cE4, I = 0.015 | label I = 15
graph NAD (high) + A110
sheet nad-a110-a.csv
column 2 | param E = 1 * cE5, I = 0.000 | label I = 0 nM
[output]
directory ./project/IMPDH/01/rates/output/compare-v
[settings]
{Filter}
XMax = 7000
LevelPercent = 90
{Output}
WriteTeX = y
ResidualsEPS = n
YAxisLabel = v, mOD/sec
[end]
A.2.3. Kinetic isotope effect

This DynaFit [2] script performs global fit of combined 1 H-IMP and 2 D-IMP data to the
steady-state rate equation that takes into account substrate inhibition by NAD+ . The enzyme
concentration is assumed to be identical in both experiments.
Fit H/D isotope effect data. Show 1H-IMP and 2D-IMP data in the
same graph, but assign to both curves unique kcat and Km values.
Use Nothrops 1983 equation where "kK = kcat/Km" is a parameter.
;______________________________________________________________________
[task]
task = fit
data = generic
[parameters]
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
[end]
A.2.4. Apparent Michaelis constant of IMP

This DynaFit [2] script used to determine the apparent Michaelis constant of IMP at [NAD+ ]
= 1.5 mM.
Fit IMP saturation data at NAD+ = 1200 to determine
the apparent Km for IMP at this NAD+ concentration.
;______________________________________________________________________
[task]
task = fit
data = generic
[parameters]
S, Vmax, Km
[model]
Vmax = 0.5 ?
Km = 50 ?
v = Vmax * S/(S + Km)
[data]
variable S
graph IMP : Km^{(app)} = (61 +/- 3) {/Symbol m}M
set rates
[output]
directory ./project/impdh/01/rates/output/fit-475
[settings]
{Output}
XAxisLabel = [IMP], {/Symbol m}M
50
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]
A.2.5. Apparent inhibition constant of XMP

This DynaFit [2] script used to determine the apparent inhibition constant of XMP at [NAD+ ]
= 1.5 mM and [IMP] = 200 M.
Determine apparent Ki for XMP at NAD+ = 1500 uM and IMP = 200 uM.
;______________________________________________________________________
[task]
task = fit
data = generic
[parameters]
P, Vo, Ki
[model]
Vo = 0.8 ?
Ki = 1000 ?
v = Vo * Ki / (P + Ki)
[data]
variable P
graph XMP : Ki^{(app)} = (1160 +/- 50) {/Symbol m}M
set rates
[output]
[settings]
{Output}
XAxisLabel = [XMP], {/Symbol m}M
ResidualsEPS = n
WriteTeX = y
IncludeYZero = y
[set:rates]
XMP rate
0 0.8958
51
125 0.8009
250 0.7491
500 0.6407
1000 0.4744
2000 0.3232
[end]
A.2.6. Apparent inhibition constant A110 in dependence on [XMP]

This DynaFit [2] script used to determine the apparent inhibition constant of A110 at various
fixed concentrations of [XMP] ([NAD+ ] = 1.5 mM and [IMP] = 200 M).
Apparent inhibition constants for A110 vs. XMP
[task]
task = fit
data = generic
[parameters]
I, E, K, Vo
[model]
E = 0.01
e = E - I - K
v = Vo*(e + sqrt(e*e + 4*E*K))/(2*E)
[data]
variable I
graph A110 : K_i^{(app)} vs. [XMP]
sheet a110-xmp.csv
column 2 | param Vo = 1 ?, K = 0.05 ? | label P =
[output]
[settings]
{Output}
IncludeYZero = y
WriteTeX = y
XAxisLabel = [I], {/Symbol m}M
ResidualsEPS = n
[end]
0 uM
125
250
500
1000
2000
A.2.7. Simulate dependence of Ki for A110 on [XMP]

This DynaFit [2] script used to simulate the expected dependence of Ki for A110 on [XMP].
[task]
task = simulate
data = generic
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 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]
A.2.8. Linear fit of simulated Ki vs. [XMP] values

This DynaFit [2] script used to perform linear fit of the simulated dependence of Ki for
A110 on [XMP]. Note that that CSV data file being pointed to is the same file that is mentioned
in Appendix A.2.7. The parameter a is the slope of the linear fit.
[task]
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 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}
ResidualsEPS = n
[end]
=
=
=
=
=
=
=
5.12
2.56
1.24
0.64
0.32
0.08
0.04 uM
A.2.9. Linear fit of experimental Ki vs. [XMP] values

This DynaFit [2] script used to perform weighted linear fit of the experimentally observed
dependence of Ki for A110 on [XMP]. The reciprocal value of the formal standard error of
individual Ki determinations is used as a weighting factor in the regression analysis.
[task]
task = fit
data = generic
[parameters]
XMP, Ki0, a
[model]
Ki0 = 0.1 ?
a = 0.0001 ?
KiApp = Ki0 + XMP * a
[data]
variable XMP
error data
sheet xmp-kiapp.csv
graph A110 : K_i^{(app)} vs. [XMP]
column 2 error 3
; error-weighted fit!
54
[output]
directory ./project/impdh/01/rates/output/fit-kiapp-001
[settings]
{Output}
IncludeYZero = y
ResidualsEPS = n
[end]
B. Raw data files and DynaFit script files

This section describes the raw experimental data files distributed with this report, as well as
the distributed input script files for the software DynaFit [1, 2] that were used to perform all
kinetic analyses. The main purpose is to allow any interested person to reproduce our kinetic
analyses and in so doing independently verify the results. A secondary goal is to provide sufficient level of detail that might allow interested parties to reproduce the kinetic results reported
here while using other third-party software packages. Both types of data files (raw data and DynaFit scripts) are collected in the ZIP archive file BaIMPDHdL-A110-transient-DynaFit.ZIP,
which represents the second of two Supporting Information components, the first component
being this PDF file.
B.1. Installation instructions
In order to reproduce or modify all data kinetic data analyses presented in this report, please
follow these steps:
1. Download the most recent version of the DynaFit software package [2] for any particular
platform (Mac OS or Windows) from the BioKin website.8
2. Install the software package simply by extracting the downloaded ZIP archive file (e.g.,
dynafit4-win.zip). This will create a directory named DynaFit4.
3. Automatically generate and install the free academic license, by filling out the online license request form. 9
4. Download and extract the Supporting Information file BaIMPDHdL-A110-transient-DynaFit.ZIP
from the ACS journal page. This will create a directory named ./project/IMPDH/01, containing a number of sub-directories as described below.
5. Move or copy the newly extracted directory ./project/IMPDH/01 into the DynaFit main
installation directory. The final layout of files and directories must match the schematic
diagram below.
\---DynaFit4
+---examples
|
+---help
|
8 http://www.biokin.com/dynafit/
9 http://www.biokin.com/dynafit/license/academic.html
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.
Start the software by double-clicking on the DynaFit program icon.

Select menu File ... Open or equivalently press Ctrl+O.
Navigate to the directory DynaFit4/project/IMPDH/01 (see above).
Navigate select the desired script file in either the progress or rates subdirectory.
For example, to repeat the kinetic analysis illustrated in Figure S9, open the
DynaFit script file .../rates/fig-S9.txt. See also Table S10.
5. Select menu File ... Run or equivalently press Ctrl+U.

Wait for the script file execution to complete. Important Note: Depending on
the type of problem, DynaFit execution can last from a few seconds to multiple
minutes of even hours. Cancel the program execution by pressing the [Cancel]
button if necessary.
6. Select menu View ... Results or equivalently press Ctrl+J.
7. Navigate in the output files using default installed web browser.
B.2. Organization of files and directories
The Supporting Information ZIP archive file contains two types of text files:
DynaFit script files (.TXT).
Raw data files (.CSV).
B.2.1. DynaFit script files
Table S10 lists the DynaFit input script files corresponding to all Figures and Tables presented
in this supporting information document as well as in the main manuscript.
Table S10 lists the DynaFit script files (file extension .TXT) distributed in the ZIP archive
file BaIMPDHdL-A110-transient-DynaFit.ZIP. All files and directories listed below are located in the main project subdirectory ./project/IMPDH/01/. Thus for example the directory
./progress/fit/ listed in the table is actually the ./project/IMPDH/01/progress/fit/ in the ZIP
archive.
Under the Microsoft Windows operating system, a convenient method to process all included
script files at once is to follow these steps:
1. Locate a particular batch file precursor file, file extension .BA_.
2. Rename the precursor file as .BAT (Windows console batch file).
56
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
Script file name(s)(a)
./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
Data File(s) (a)
./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
a110-xmp
N1H1I1
N1H1I1
N1H1I1
NxHyIz (b)
NxHyIz (b)
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

Inhibition of Inosine-5 - Monophosphate Dehydrogenase From Bacillus Anthracis: Mechanism Revealed by Pre-Steady-State Kinetics

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Inhibition of Inosine-5 - Monophosphate Dehydrogenase From Bacillus Anthracis: Mechanism Revealed by Pre-Steady-State Kinetics

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Article

Inhibition of Inosine-5-monophosphate Dehydrogenase from

Department of Biology, Brandeis University, Waltham, Massachusetts 02454, United States

Department of Chemistry, Brandeis University, Waltham, Massachusetts 02454, United States

ABSTRACT: Inosine-5-monophosphate dehydrogenase

of these compounds are also eective against IMPDHs from

ntibiotic resistance is a worldwide problem threatening the

Received: March 23, 2016

the adjustable baseline oset at 0.004 s, essentially a property of

MATERIALS AND METHODS

([E]0 [I]0 K i*)2 + 4[E]0 K i*

where square brackets with lower index zero represent total or

where A is the absorbance (in milli-optical density units, 103

bounds), are fully determined by the available experimental

RESULTS AND DISCUSSION

Figure 1. Representative data set from the global t of stopped-ow

concentration of EAB (blue dashed curve). The two

Scheme 2. Minimal Kinetic Mechanism of BaIMPDH

Figure 2. Evolution of enzyme species concentrations corresponding

interval is not dened at the 5% SSQ condence level, which

determined uniquely in terms of their well-reproduced best-t

and 1000 M1 s1 (kon). The purpose was to determine the

tting a single data set, compared to a kinetic mechanism with

the observed reaction rate (approximately 0.4 10 3

Steady-State Initial Rate Kinetics. Predicted versus

validate this prediction, the combined initial rate data were

variable NAD+ concentrations was reported to be 58 4 nM13

associated formal standard errors from nonlinear regression are

similar to that found in IMPDHs from from many pathogenic

According to Scheme 3, this compound binds preferentially

The Supporting Information is available free of charge on the

(1) World Health Organization. Antimicrobial resistance: Global

*E-mail: hedstrom@brandeis.edu. Phone: +1 (781) 736-2333.

Y.W.: Sanford-Burnham-Prebys Medical Discovery Institute,

G.M.: Department of Pharmaceutics, Indian Institute of

This work was supported by National Institutes of Health

The authors declare no competing nancial interest.

(41) Rawlings, J. O. (1988) Applied Regression Analysis: A Research

(17) Maurya, S. K., Gollapalli, D. R., Kirubakaran, S., Zhang, M.,

Inhibition of IMPDH from Bacillus anthracis. Mechanism

of Biology and Chemistry, Brandeis University, Waltham, Massachusetts

3 Steady-state initial rate kinetics

Biochemistry Ms. No bi-2016-00265u.R2

3.2.1 Classical rate equation (King-Altman & Cleland)

B Raw data files and DynaFit script files

new batch of NAD+

Table S1: Organization of replicated stopped-flow experiments. Replicate labels correspond to

Table S2: Combinatorial replication scheme. Each of N, H, and I replicates consists of

Regression equation and model parameters

k2 [EA][B] + k2 [EAB] + k5 [EP] k8 [EA][I] + k8 [EAI]

k2 [EA][B] + k2 [EAB] k7 [EP][B] + k7 [EPB]

+k2 [EA][B] k2 [EAB] k3 [EAB] + k3 [EPQ]

+k3 [EAB] k3 [EPQ] k4 [EPQ] + k4 [EP][Q]

+k4 [EPQ] k4 [EP][Q] k5 [EP] k7 [EP][B] + k7 [EPB]

+k4 [EPQ] k4 [EP][Q]

+k7 [EP][B] k7 [EPB]

k8 [EA][I] + k8 [EAI] k9 [EP][I] + k9 [EPI]

+k8 [EA][I] k8 [EAI]

+k9 [EP][I] k9 [EPI]

applies to trace no.

Initial estimates of model parameters

Empirical confidence intervals

empirical interval, 5 % SSQ increase

([E]0 [I]0 K i)2 + 4[E]0 K i