Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5′-phosphate decarboxylase-catalyzed reactions

Article abstract of DOI:10.1021/bi401117y

The side chain cation of Arg235 provides a 5.6 and 2.6 kcal/mol stabilization of the transition states for orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC) from Saccharomyces cerevisiae catalyzed reactions of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP), respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-like transition state for enzyme-catalyzed exchange of the C-6 proton of 5-fluorouridine 5′-monophosphate (FUMP), but no stabilization of the transition states for enzyme-catalyzed decarboxylation of truncated substrates 1-(β-d- erythrofuranosyl)orotic acid and 1-(β-d-erythrofuranosyl) 5-fluorouracil. These observations show that the transition state stabilization results from formation of a protein cation-phosphodianion pair, and that there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/mol side chain interaction with the transition state for the decarboxylation reaction is 50% of the total 11.2 kcal/mol transition state stabilization by interactions with the phosphodianion of OMP, whereas the 7.2 kcal/mol side chain interaction with the transition state for the deuterium exchange reaction is a larger 78% of the total 9.2 kcal/mol transition state stabilization by interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange is expressed predominantly as a change in the turnover number k _ex, whereas the effect on the enzyme-catalyzed decarboxylation of OMP is expressed predominantly as a change in the Michaelis constant K_m. These results are rationalized by a mechanism in which the binding of OMP, compared with that for FUMP, provides a larger driving force for conversion of OMPDC from an inactive open conformation to a productive, active, closed conformation.

Full text of DOI:10.1021/bi401117y

Article
pubs.acs.org/biochemistry
Role of a Guanidinium Cation−Phosphodianion Pair in Stabilizing
the Vinyl Carbanion Intermediate of Orotidine 5′-Phosphate
Decarboxylase-Catalyzed Reactions
†
†
†
‡
,†
Bogdana Goryanova, Lawrence M. Goldman, Tina L. Amyes, John A. Gerlt, and John P. Richard*
†
Department of Chemistry, University at Buffalo, Buffalo, New York 14260, United States
‡
Department of Biochemistry and Chemistry, University of Illinois, Urbana, Illinois 61801, United States
*
S Supporting Information
ABSTRACT: The side chain cation of Arg235 provides a 5.6
and 2.6 kcal/mol stabilization of the transition states for
orotidine 5′-monophosphate (OMP) decarboxylase
(
OMPDC) from Saccharomyces cerevisiae catalyzed reactions
of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP),
respectively, a 7.2 kcal/mol stabilization of the vinyl carbanion-
like transition state for enzyme-catalyzed exchange of the C-6
proton of 5-fluorouridine 5′-monophosphate (FUMP), but no
stabilization of the transition states for enzyme-catalyzed
decarboxylation of truncated substrates 1-(β-D-erythrofuranosyl)orotic acid and 1-(β-D-erythrofuranosyl) 5-fluorouracil. These
observations show that the transition state stabilization results from formation of a protein cation−phosphodianion pair, and that
there is no detectable stabilization from an interaction between the side chain and the pyrimidine ring of substrate. The 5.6 kcal/
mol side chain interaction with the transition state for the decarboxylation reaction is 50% of the total 11.2 kcal/mol transition
state stabilization by interactions with the phosphodianion of OMP, whereas the 7.2 kcal/mol side chain interaction with the
transition state for the deuterium exchange reaction is a larger 78% of the total 9.2 kcal/mol transition state stabilization by
interactions with the phosphodianion of FUMP. The effect of the R235A mutation on the enzyme-catalyzed deuterium exchange
is expressed predominantly as a change in the turnover number k , whereas the effect on the enzyme-catalyzed decarboxylation
ex
of OMP is expressed predominantly as a change in the Michaelis constant K . These results are rationalized by a mechanism in
m
which the binding of OMP, compared with that for FUMP, provides a larger driving force for conversion of OMPDC from an
inactive open conformation to a productive, active, closed conformation.
The mechanism of action of orotidine 5′-monophosphate
decarboxylase (OMPDC) has attracted the interest of
enzymologists and other chemists interested in catalysis
50/50 H O/D O, in protonation of the vinyl carbanion is
2 2
consistent with a short lifetime for the reaction intermedi-
1
6,17
ate.
17
because of the difficulty in rationalizing the enormous 10 -
fold enzymatic rate acceleration of chemically difficult non-
enzymatic decarboxylation of orotidine 5′-monophosphate
OMPDC provides a large ∼31 kcal/mol stabilization of the
transition state for the decarboxylation of OMP and a smaller
1
∼
8 kcal/mol stabilization of the Michaelis complex. These
(
OMP, Scheme 1A, X = H) to give uridine 5′-monophosphate
observations show that it is necessary to determine both the
network of stabilizing protein−transition state interactions and
the mechanism by which these interactions develop on moving
from the Michaelis complex to the reaction transition state. The
interactions between OMPDC and the substrate phosphodian-
ion provide an 11.2 kcal/mol stabilization of the transition state
1,2
(UMP). The well-known large barrier to nonenzymatic
decarboxylation of OMP, which arises from the great instability
1
,3−5
of the putative vinyl carbanion reaction intermediate,
prompted proposals of reaction mechanisms that avoid
6
−12
formation of this intermediate
but surprisingly, little
experimental data in support of any mechanism, until it was
shown that OMPDC catalyzes both the decarboxylation of
OMP and 5-fluoroorotidine 5′-monophosphate (FOMP,
Scheme 1A, X = F) and exchange of the C-6 proton of UMP
and of 5-fluorouridine 5′-monophosphate (FUMP) for
for decarboxylation, which is 40% of the total transition state
18,19
stabilization.
Interactions between OMPDC and the
phosphodianion of 5-fluorouridine 5′-monophosphate
(
FUMP) provide a smaller 9.2 kcal/mol stabilization of the
13−15
transition state for the enzyme-catalyzed deuterium exchange
deuterium from solvent D O (Scheme 1B).
These results
2
20
reaction. This intrinsic phosphodianion binding energy is
provide compelling evidence that OMPDC-catalyzed reactions
are through a vinyl carbanion reaction intermediate that is
strongly stabilized by interactions with the protein catalyst. The
unusual failure of OMPDC to differentiate between the
reaction of hydrogen and deuterium, from a mixed solvent of
Received: August 15, 2013
Revised: September 20, 2013
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/bi401117y | Biochemistry XXXX, XXX, XXX−XXX

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Article
Scheme 1
similar to that determined for the proton and hydride-ion
transfer reactions, catalyzed by triosephosphate isomerase and
α-glycerol phosphate dehydrogenase, respectively.
reaction of 1-(β-D-erythrofuranosyl)-5-fluorouracil (FEU)
18,20
(Scheme 2).
This is the result of utilization of protein−
21−24
dianion binding interactions to activate OMPDC for catalysis of
reactions at the distant pyrimidine ring.
The 9−11 kcal/mol phosphodianion binding energy might
be utilized to anchor the substrate to OMPDC (K effect) or to
The X-ray crystal structure of OMPDC from Saccharomyces
cerevisiae (ScOMPDC) liganded with 6-hydroxyuridine 5′-
monophosphate (Figure 1) shows that the phosphodianion
m
activate the enzyme for catalysis of the decarboxylation and
deuterium exchange reactions in which case the binding
interactions will be expressed specifically at the transition
25
state for the enzyme-catalyzed reaction (kcat effect). We have
distinguished between anchoring and activating interactions of
OMPDC with the phosphodianion of OMP or FUMP by
cutting the covalent connection that anchors the phosphodian-
ion to the whole substrates and examining the OMPDC-
catalyzed reactions of substrate pieces (Scheme 2). The binding
2
−
of the phosphite dianion (HPO3 ) to OMPDC results in a
large increase in the observed second-order rate constant
k /K for enzyme-catalyzed decarboxylation of 1-(β-D-
cat
m
erythrofuranosyl)orotic acid (EO) and the deuterium exchange
Scheme 2
Figure 1. X-ray crystal structure of yeast OMPDC liganded with 62-
hydroxyuridine 5′-monophosphate (Protein Data Bank entry 1DQX).
The structure shows the interactions between the side chains of
Gln215, Tyr217, and Arg235 of the protein with the bound
phosphodianion group. Reprinted with permission from ref 19.
Copyright 2012, American Chemical Society.
group of the ligand interacts directly with the side chains of
Gln215 and Tyr217, which are part of a closed flexible loop,
2
and with the guanidinium group of Arg235. The focus of this
paper is the strong ion-pairing interaction between the
phosphodianion and the guanidinium cation side chain of
Arg235 that accounts for 50% of the total intrinsic
26,27
phosphodianion binding energy.
We are interested in
determining whether these interactions are expressed at the
transition state for the OMPDC-catalyzed deuterium exchange
reaction of FUMP, in which case they would be utilized mainly
in the stabilization of the vinyl carbanion intermediate common
to the decarboxylation and deuterium exchange reactions.
We report the effect of the R235A mutation of ScOMPDC
on the kinetic parameters for the following enzymatic reactions:
(
1) the enzyme-catalyzed deuterium exchange reaction of
FUMP, (2) the rescue, by guanidinium cation, of the activity of
B
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the R235A mutant of OMPDC for catalysis of the deuterium
exchange reaction of FUMP, and (3) the enzyme-catalyzed
decarboxylation of FOMP and of the truncated substrate 1-(β-
D-erythrofuranosyl) 5-fluoroorotic acid (FEO). These data are
analyzed along with results from earlier studies of the effect of
the R235A mutation on OMPDC-catalyzed decarboxylation of
1-(β-D-Erythrofuranosyl) 5-fluoroorotic acid (FEO) was
28
synthesized as described in earlier work. 5-Fluoroorotidine
5′-monophosphate (FOMP) was synthesized enzymatically by
adaptation of the procedure for the enzymatic synthesis of
35
UMP. A typical reaction mixture (10 mL, 30 °C) contained,
initially, 0.25 mmol of 5-fluoroorotic acid, 4 units of PRPP
synthase, 20 units of ORTase, 200 units of adenylate kinase,
1
9,28
OMP and EO.
2
00 units of pyruvate kinase, 300 units of yeast inorganic
EXPERIMENTAL SECTION
pyrophosphatase, 50 mM MgCl , 3 mM ATP, 60 mM PEP, and
■
2
3
mM DTT in 30 mM phosphate buffer at pH 7.5. The
Materials. D-Ribose 5-phosphate (disodium salt ≥98%), 5-
formation of FOMP was monitored in aliquots that were
diluted 250-fold into an assay mixture for OMPDC by
determining the decrease in absorbance at 282 nm from
OMPDC-catalyzed decarboxylation of FOMP. The yield of
FOMP plateaued after 90 min, at which time an additional 0.25
mmol of 5-fluoroorotic acid was added. The reaction was
monitored for an additional 150 min, when the yield of FOMP
again plateaued. The enzymes were removed by filtration using
an Amicon Ultra-15 10 KDa MWCO membrane centrifugal
filter, and the FOMP was purified by ion exchange
chromatography over DEAE Sephadex A25 and eluting with
a gradient of triethylammonium bicarbonate. The fractions that
contained FOMP were pooled, and the buffer salts were
removed on a rotary evaporator at reduced pressure followed
by lyophilization. The lyophilizate was suspended in 25 mL o+f
methanol that contained 25 g of Amberlite IR120 resin (H
form). The solution was stirred for 1 h at room temperature
with gentle purging of Ar. The resin was then removed by
filtration, washed with 200 mL of methanol, and the solvent
was removed under reduced pressure. The compound was
dissolved in water, and colored material from the Amberlite
resin was removed by passage through a 3 mL Supelco reversed
phase (LC-18) SEP tube. This gave the free acid of FOMP as a
colorless solid in 40% yield.
fluorouridine (99%), phosphoryl chloride (99%), phosphoe-
nolpyruvate (monosodium salt, 98%), adenosine 5′-triphos-
phate (disodium salt, 99%), β-nicotinamide adenine dinucleo-
tide (disodium salt, 98%), and triethylamine (99.5%) were
purchased from Sigma. 3-(N-Morpholino)propanesulfonic acid
(
99.5%) and trimethyl phosphate (97%) were purchased from
Fluka, imidazole (99.5%), guanidinium hydrochloride (99%),
and potassium phosphate (99.8%) were from Fisher. Ultrapure
glycylglycine (>99%) was obtained from USB Biochemicals.
Dowex-50 (hydrogen form), DEAE Sephadex A-25, and
Amberlite IR120 ion exchange resins were purchased from
Sigma. The following deuterium labeled compounds were
purchased from Cambridge Isotope Laboratories: D O
2
(
9
99.9%), DCl (35 wt %, 99.9% D), and NaOD (30 wt %,
8% D). The water was distilled and purified in a Milli-Q water
purification system. The imidazole was recrystallized from
benzene and dried under vacuum at room temperature for
several days. Trimethyl phosphate was dried over activated
molecular sieves (4 Å) and distilled under reduced pressure. 5-
Fluorouridine was dried at 50 °C under reduced pressure for 24
h prior to use. All other reagents were used without further
purification.
L-Lactate dehydrogenase, inorganic pyrophosphatase, adeny-
late kinase, and pyruvate kinase were purchased from Sigma.
Orotate phosphoribosyl transferase (ORTase) from Salmonella
typhimurium was overexpressed in Escherichia coli BL21 (DE3)
transformed with the plasmid pOPRTase. The isolated
OPRTase was purified according to a literature procedure.
Phosphoribosylpyrophosphate (PRPP) synthetase from E. coli
was constitutively expressed from E. coli strain HO1702
harboring the plasmid pHO11 that was a generous gift from
NMR Analyses. 19F NMR spectra were acquired on a
Varian Unity Inova-500 spectrometer operating at 470 MHz.
Spectra (64 transients), obtained to monitor enzyme-catalyzed
deuterium exchange reactions, were recorded using a spectral
width of 50 000 Hz, a 90° pulse angle, a 2.6 s acquisition time,
29
30
20
and a relaxation delay between pulses of 28 s (>7T ). The
1
chemical shifts of the 19F NMR signals are referenced to the
value of −78.5 ppm for a neat trifluoroacetic acid external
standard.
3
1,32
Professor Vern Schramm
literature procedure. Published procedures were followed to
and was purified according to a
33
prepare wild-type OMPDC from Saccharomyces cerevisiae
Preparation of Solutions. The solution pH was
determined at 25 °C using an Orion Model 720A pH meter
equipped with a Radiometer pHC4006-9 combination
electrode that was standardized at pH 7.00 and 10.00 at 25
27
(
ScOMPDC) and the R235A mutant of ScOMPDC.
Orotidine 5′-monophosphate was prepared by enzymatic
34
methods from orotate and phosphoribosylpyrophosphate.
This synthesis gave OMP that contained ammonium
bicarbonate from purification by column chromatography that
was removed by the following procedure. Stock solutions of
OMP (∼10−25 mM) were carefully titrated to pH 5 with 1.00
°C. The pD of the buffers prepared in D O was obtained by
2
36
adding 0.4 to the reading on the pH meter. The acidic
protons of GlyGly were exchanged for deuterium by dissolving
the buffer in D O followed by evaporation and drying under
2
M HCl, and N was then bubbled through the solution to carry
vacuum at 55 °C. Buffered solutions of GlyGly were prepared
2
away CO . The solution pH was adjusted to 7.1 and monitored
by dissolving the buffer acid in D O and adjusting to the
2
2
for several hours. If an increase in pH was observed due to the
required pD using 0.98 M NaOD. The concentration of FOMP
and FUMP in 0.1 M HCl was determined from the absorbance
evaporation of CO , then additional 1.00 M HCl was added
2
−1
−1
−1
and the bubbling of nitrogen repeated. The concentration of
at 271 nm (ε = 10200 M cm ) and 270 nm (ε = 9160 M
13,28
−1
NH Cl in the final NH CO free solution was determined as
cm ), respectively.
Samples of R235A mutant ScOMPDC
4
4
3
−
the concentration of HCl used to convert the HCO3 to CO2.
The concentration of this salt was included in the calculation of
the solution ionic strength.
that had been stored at −80 °C were defrosted and dialyzed at
7 °C against 50 mM MOPS (45% free base) at pH 7.0 and I =
0.14 (NaCl) for the experiments with FEO. Samples of wild-
type ScOMPDC were dialyzed at 7 °C against 10 mM MOPS at
pH 7.1 and I = 0.10 (NaCl). Samples of R235A mutant
ScOMPDC used to study catalysis of the deuterium exchange
The triethylammonium salt of FUMP was synthesized as
13
described in earlier work and was converted to the free acid
+
by passage over Amberlite IR120 resin (H -form) in methanol.
C
dx.doi.org/10.1021/bi401117y | Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
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reaction were dialyzed at 7 °C against 10 mM MOPS at pH 7.1
and I = 0.10 (NaCl). This was followed by dialysis against
several changes of 60 mM GlyGly (pD 8.15) at I = 0.14 (NaCl)
velocity of the enzyme-catalyzed deuterium exchange reaction
was examined, reactions in a volume of 1−2 mL were initiated
by addition of an aliquot of h-FUMP in D O to a mixture of 50
2
in D O using a D-tube dialyzer (10 kDa MWCO, Novagen)
mM GlyGly at pD 8.15, NaCl, and ScOMPDC in D O to give
2
2
placed inside a narrow vessel that was isolated from
atmospheric moisture using parafilm. The concentration of
stock solutions of wild-type and R235A mutant ScOMPDC was
determined from the absorbance at 280 nm by using an
final concentrations of 0.74 mM h-FUMP, 12.5−82 mM
guanidinium cation, and 0.08−0.12 mM ScOMPDC at I = 0.14.
At timed intervals, during each set of experiments, 20 μL of
neat deuterium labeled formic acid (DCOOD) was added to a
measured aliquot that contains 0.38 or 0.75 μmol of h-FUMP +
d-FUMP to quench the reaction. The enzyme was removed by
ultracentrifugation using an Amicon Ultrafiltration device (10
KDa MWCO), the volume of the filtrate was adjusted to 700
−1
−1
extinction coefficient of 29 900 M cm , calculated with the
37
ProtParam tool available on the ExPASy server.
Steady-State Enzyme Kinetics. The decarboxylation of
OMP catalyzed by OMPDC was monitored spectrophoto-
metrically by following the decrease in absorbance at 279, 290,
μL with D O, and the solution was transferred to an NMR tube
2
−
1
−1
or 295 nm [Δε, λ: −2400 M cm , 279 nm; −1620 M cm
for analysis of the formation of d-FUMP. The enzyme-
catalyzed deuterium exchange reactions were monitored for up
to 2 days, during which time the activity of ScOMPDC was
determined by a standard assay. In several cases, the enzyme
was removed after completion of the reaction by the filtration
procedures described above and the solution pD was
determined. No significant loss in enzymatic activity (<5%)
or decrease in solution pD (<0.1 unit) was observed during the
course of these enzyme-catalyzed deuterium exchange reac-
tions.
−1
2
90 nm; −842 M cm , 295 nm]. These wavelengths were
chosen so that the initial absorbance A were ≤2.0. Assays (1
i
mL) contained OMP (0.05−1.5 mM) at pH 7.1 (30 mM
MOPS), 25 °C, and at I = 0.10 maintained with NaCl. The
reactions were initiated by the addition of a stock solution of
R235A mutant ScOMPDC to give a final enzyme concentration
−1
of 1 μM. The initial velocity v (M s ) was determined by
i
monitoring the decrease in absorbance at the chosen
wavelength. Values of kcat and Km were obtained from the
19
nonlinear least-squares fits of seven or more values of v /[E]
The F NMR spectra were determined to give the integrated
peak areas for the doublet of doublets with area (A ) at
−165.36 ppm for h-FUMP and the broad upfield-shifted
apparent doublet with area (A ) at −165.66 ppm for d-FUMP.
The initial velocity (v , eq 1) for the enzyme-catalyzed
deuterium exchange reactions of h-FUMP was determined as
the slopes of the linear plots of f [FUMP] against time over
the first 5−10% reaction, where f = A /(A + A ) is the
fraction of total FUMP labeled with deuterium at the C-6
position and [FUMP] is the initial concentration of h-FUMP.
i
−1
(
s ) to the Michaelis−Menten equation.
H
Assays of R235A mutant ScOMPDC-catalyzed decarbox-
ylation of FOMP contained FOMP (0.10−1.0 mM) at pH 7.1
D
(
10 mM MOPS), 25 °C, and I = 0.10 maintained with NaCl.
i
−1
Initial velocities, ν (M s ), of the decarboxylation of FOMP
i
catalyzed by R235A mutant ScOMPDC were determined
P
o
spectrophotometrically by monitoring the decrease in absorb-
P
D
H
D
−1
−1
ance at 290 nm (Δε = −1090 M cm ). Assays in a volume of
1
mL were initiated by the addition of 1 μL of a stock solution
o
of R235A mutant ScOMPDC to give a final enzyme
⎛
⎝
d{f [FUMP] } ⎞
o
P
concentration of 15−20 nM. Values of k and Km for the
cat
v = ⎜
⎟
⎠
i
R235A ScOMPDC-catalyzed decarboxylation reaction of
FOMP were obtained from the nonlinear least-squares fit of
dt
(1)
the values of ν /[E] to the Michaelis−Menten equation.
i
RESULTS
■
R235A ScOMPDC-Catalyzed Decarboxylation of FEO.
The decarboxylation of FEO (0.14 mM) catalyzed by R235A
mutant ScOMPDC (30 μM) at pH 7.0 (25 mM MOPS), 25
The decarboxylation of OMP catalyzed by the R235A mutant
of ScOMPDC was monitored spectrophotometrically at
wavelengths chosen to ensure that A < 2.0 for the initial
absorbance of the OMP. Figure 2 shows the dependence of the
initial velocity v /[E] on [OMP] for decarboxylation reactions
catalyzed by the R235A mutant of OMPDC at 25 °C, pH 7.1
30 mM MOPS), and at different ionic strengths maintained
°
C, and an ionic strength of 0.14 (NaCl) was followed
i
spectrophotometrically by monitoring the decrease in absorb-
ance at 282 nm, as described in a previous study using wild-type
i
2
8
ScOMPDC. These reactions were monitored for ≥10
reaction half-times (≤800 min), during which time ScOMPDC
was shown by a standard enzyme assay to maintain full catalytic
activity. Observed first-order rate constants kobs (s ) for
enzyme-catalyzed decarboxylation of FEO were obtained from
the fits of reaction progress curves to an equation for a single
(
with NaCl. Table S1 (Supporting Information) reports the
−1
18
exponential decay. Second-order rate constants (k /K )
cat
m obs
were obtained using the relationship (k /K ) = k /[E].
cat
m obs
obs
R235A ScOMPDC-Catalyzed Deuterium Exchange at
C-6 of h-FUMP Monitored by 19F NMR Spectroscopy.
The C-6 deuterium exchange reactions of h-FUMP catalyzed
by the R235A mutant of ScOMPDC in D O at pD 8.15 were
2
19
monitored by F NMR spectroscopy at 470 MHz. Reactions in
a volume of 1−2 mL were initiated by the addition of an aliquot
of h-FUMP in D O to a mixture of 50 mM GlyGly (20% free
base) at pD 8.15, NaCl, and the R235A mutant of ScOMPDC
2
Figure 2. Michaelis−Menten plots that show the dependence of v /[E]
i
for decarboxylation of OMP catalyzed by R235A mutant ScOMPDC
on the concentration of OMP for reactions at 25 °C, pH 7.1 (30 mM
MOPS) and at different ionic strengths maintained with NaCl: ●, I =
0.10; ▲, I = 0.050; ▼, I = 0.035; ◆, I = 0.020.
in D O to give final concentrations of 0.74−9.6 mM h-FUMP
2
and of 0.2−0.9 mM mutant ScOMPDC at I = 0.14. In
experiments where the effect of guanidinium cation on the
D
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Biochemistry
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individual values of v /[E] and the wavelengths monitored for
activation of R235A ScOMPDC-catalyzed decarboxylation and
deuterium exchange reactions. The protocol to monitor R235A
mutant ScOMPDC-catalyzed deuterium exchange reactions of
FUMP is labor intensive and consumes large amounts of
enzyme. This prohibited the determination of kinetic
parameters for activation of the mutant enzyme-catalyzed
i
these experiments. The solid lines for Figure 2 show the
nonlinear least-squares fits of the experimental data to the
Michaelis−Menten equation. The following values of k , K ,
cat
m
and k /K for decarboxylation of OMP catalyzed by R235A
cat
m
mutant ScOMPDC were obtained from these fits, where the
quoted uncertainties represent the 95% confidence interval: ●,
+
deuterium exchange reaction by Gua for comparison with
−
1
−3
+
I = 0.10, kcat = 1.0 ± 0.19 s , K = (1.1 ± 0.36) × 10 M,
previously published kinetic parameters for Gua activation of
m
−1
−1
−1
27
k /K = 910 M s ; ▲, I = 0.050,k = 0.93 ± 0.11 s , K
mutant enzyme-catalyzed decarboxylation of OMP.
cat
=
0
cat
m
cat
m
−
3
−1 −1
(0.23 ± 0.065) × 10 M, k /K = 4000 M s ; ▼, I =
cat m
1
−
−3
.035, kcat = 1.21 ± 0.15 s , K = (0.23 ± 0.052) × 10 M,
m
1
−
−1
−1
k /K = 5300 M s ; ◆, I = 0.020, k = 1.33 ± 0.10 s ,
m
cat
−
3
−1 −1
Km = (0.16 ± 0.022) × 10 M, k /K = 8300 M s .
cat
m
The R235A mutant ScOMPDC-catalyzed deuterium ex-
change reactions of h-FUMP in solutions buffered by 50 mM
1
9
GlyGly in D O at pD 8.15 and 25 °C were monitored by
F
2
NMR spectroscopy at 470 MHz, as described in an earlier study
of the wild-type enzyme-catalyzed deuterium exchange
2
0
reactions of 1-(β-D-erythrofuranosyl) 5′-fluorouracil (FEU).
The initial reaction velocities (v ) were determined as described
i
in the Experimental Section. The dependence of v /[E] on the
i
concentration of the substrate is shown in Figure 3. These data
were fit to eq 2, derived for Scheme 3, to give the values for kex
and K reported in Table 1.
Figure 4. Dependence of (v )obs/(v ) on concentration of guanidinium
i i o
d
cation for R235A mutant ScOMPDC-catalyzed reactions, where (v ) is
i
o
the observed initial velocity in the absence of the cation activator: ●,
vi
kex[h‐FUMP]o
=
the deuterium exchange reaction of 0.74 mM h-FUMP catalyzed by
the R235A mutant of ScOMPDC at pD 8.15 (20 mM GlyGly, 20%
free base), 25 °C and I = 0.14 (NaCl); ■, the decarboxylation reaction
[E]
[h‐FUMP] + Kd
(2)
o
of 5 μM OMP at pH 7.1 (10 mM MOPS), 25 °C and I = 0.10
2
7
(NaCl).
The initial velocity of R235A mutant ScOMPDC-catalyzed
decarboxylation of FOMP at 25 °C was determined for
reactions at several concentrations of FOMP between 0.10 and
1
.0 mM in solutions buffered by 10 mM MOPS at pH 7.1 and
at I = 0.10 (NaCl). Values of kcat and K for R235A mutant
m
enzyme-catalyzed decarboxylation of FOMP were obtained
from the nonlinear least-squares fits of the values of ν /[E] to
i
the Michaelis−Menten equation (Table 1). The following
parameters for ScOMPDC-catalyzed reactions, determined in
earlier work at pH 7.1 and 25 °C, are reported in Table 1: kcat
Figure 3. Dependence of v /[E] on the concentration of substrate h-
i
FUMP for the deuterium exchange reaction catalyzed by the R235A
mutant of ScOMPDC at pD 8.15 (20 mM GlyGly, 20% free base), and
I = 0.14 (NaCl). The solid line shows the correlation of the
experimental data to eq 2 derived for Scheme 3, using the values for
the kinetic parameters reported in Table 1.
and K for wild-type ScOMPDC-catalyzed decarboxylation of
m
38
39
OMP and FOMP; kex and K for the wild-type enzyme-
catalyzed deuterium exchange reaction of h-FUMP.
d
13
Figure 5 shows the increase, at increasing concentrations of
phosphite dianion, in the apparent second-order rate constant
Scheme 3
(
k /K ) for R235A mutant ScOMPDC-catalyzed decarbox-
cat m obs
ylation of FEO at pH 7.0 (25 mM MOPS), 25 °C, and an ionic
strength of 0.14 (NaCl). A linear least-squares fit of these data
2
−
Figure 4 shows the effect of increasing concentrations of
guanidinium cation (Gua ) on the relative initial velocity,
to eq 3, derived for Scheme 4 (K ≫ [HPO3 ]), gives the
d
+
values of (k /K ) and [(k /K )
]/K reported in Table
cat
m E
cat
m E·HPi
d
(
v ) /(v ) , for the R235A mutant ScOMPDC-catalyzed
i obs i o
2
. Table 2 also reports these kinetic parameters for wild-type
deuterium exchange reaction of 0.74 mM h-FUMP at 25 °C
in solutions buffered by 50 mM GlyGly at pD 8.15 and an ionic
strength of 0.14 (NaCl), and the decarboxylation reaction of 5
μM OMP reported in an earlier study. In each case, the
substrate concentration [h-FUMP] ≪ K = 5 mM or [OMP]
ScOMPDC-catalyzed decarboxylation of FEO and for wild-type
and R235A mutant ScOMPDC-catalyzed decarboxylation of
1
9,28
EO, determined in earlier studies.
o
d
o
[HPO32
−
]
kcat
⎛
+ ⎜
⎝
⎞⎛
⎞
⎟
≪
K = 1 mM is far below saturation, so that in the absence of
⎛ k
⎞
⎟
⎛ k
⎞
⎟
d
cat
cat
+
=
⎟
⎜
⎜
⎜
Gua the enzyme exists mainly in the unliganded form. The
⎝
K ⎠
⎝ K ⎠
Kd
⎠⎝ K ⎠
+
m
m
m
(3)
data in Figure 4 show that Gua provides quantitatively similar
obs
E
E·HPi
E
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Biochemistry
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Table 1. Kinetic Parameters for the Decarboxylation and Deuterium Exchange Reactions Catalyzed by Wild-Type and R235A
a
ScOMPDC
kcat or kex (s−1)
wild type R235A
Km or K (M)
k /K or k /K (M s−1
−1
)
d
cat
m
ex
d
wild type
R235A
wild type
R235A
b
1.4 × 10−6
1.1 × 10−3
1.1 × 107
OMP
15
1.0
910
effect of mutation
15-fold decrease
95
790-fold increase
8 × 10−6
73-fold increase
1.1 × 10−4
12000-fold decrease
1.2 × 107
75-fold decrease
400
c
5.8 × 10−4
5.0 × 10−3
1.6 × 105
FOMP
92
effect of mutation
no effect
d
9.3 × 10−6
1.9 × 10−3
h-FUMP
0.044
effect of mutation
4700-fold decrease
45-fold increase
210000-fold decrease
a
b
Kinetic parameters kcat and K , k and K , respectively, are used for the ScOMPDC-catalyzed decarboxylation and deuterium exchange reactions.
m
ex
d
c
At pH 7.1 (30 mM MOPS), 25 °C, and I = 0.10 (NaCl). Data for wild-type ScOMPDC is from ref 38. At pH 7.1 (10 mM MOPS), 25 °C, and I =
d
0
.10 (NaCl). Data for wild-type ScOMPDC is from ref 39. At pD 8.15 (20 mM GlyGly, 20% free base), and I = 0.14 (NaCl). Data for wild-type
ScOMPDC is from ref 13.
Scheme 4
DISCUSSION
■
The 7-fold increase in Km (Figure 2) for decarboxylation of
OMP catalyzed by the R235A mutant of ScOMPDC as the
−3
ionic strength is increased from 0.020 (K = 0.16 × 10 M) to
m
.10 (Km = 1.1 × 10− M) shows that great care must be
3
0
exercised to maintain constant ionic strength in these
experiments. A similar effect of changing ionic strength on
K has been reported for triosephosphate isomerase (TIM)-
m
40
Table 2. Kinetic Parameters from Scheme 4 for
catalyzed isomerization of glyceraldehyde 3-phosphate. This
increase in Km is consistent with a decrease in the activity
coefficient for the substrate phosphodianion at increasing ionic
strength. The ScOMPDC-catalyzed deuterium exchange
reactions of h-FUMP were carried at pD 8.15, because this is
very close to the pH optimum of k /K for the deuterium
Decarboxylation Reactions of Substrate Pieces Catalyzed by
a
Wild-Type and R235A Mutant ScOMPDC
41
EO + HPO32
−
FEO + HPO32−
ex
d
(
kcat/Km)
(kcat/Km)
Kd
(M−2 s−1
4.8 × 104
250 ± 15
E·HPi
E·HPi
exchange reactions catalyzed by wild-type ScOMPDC. Similar
K
s
d
6
7
−1 −1
(
k /K )
(M−2
−1
(k /K )
values of k /Km = 5.5 × 10 and 1.1 × 10 M
cat
s
were
cat
(M
m
s
E
)
cat
(M
m
s
E
)
−
1
−1
)
−1 −1
ScOMPDC
)
determined for wild-type ScOMPDC-catalyzed decarboxylation
b
4
20
wild type
0.026
0.026
1.2 × 10
10
12
of OMP at pD = 8.15 and at pH 7.1, respectively.
c
c
R235A
0.63
Role of Arg235 in ScOMPDC-Catalyzed Decarboxyla-
tion and Deuterium Exchange Reactions. Table 3
summarizes the effects of the R235A mutation on the
a
Second-order rate constants reported in this table are reproducible to
better than ±10%. Data for the wild-type ScOMPDC-catalyzed
reactions of EO and FEO are from refs 19 and 28, respectively.
b
second-order rate constants k /Km for several ScOMPDC-
cat
c
Reference 19.
catalyzed reactions and the stabilizing interaction between the
side chain cation and the different reaction transition states.
The essentially full 13C kinetic isotope effect on k /K for
cat
m
ScOMPDC-catalyzed decarboxylation of OMP is consistent
with the expression of the entire side chain interaction at the
34
rate-determining transition state. However, it has been
determined that the rate constant k−d for the breakdown of
E·OMP to E + OMP is only 4-fold larger than k for turnover
cat
4
2
of OMP, so that substrate binding is weakly rate determining
2
9,42
for the second-order rate constant k /K .
that k /K = 1.1 × 10 M
We calculate
cat
m
7
−1 −1
s
(Table 1) is 20% smaller than
cat
m
that for a hypothetical reaction where OMP binds reversibly
and the chemistry is rate determining for decarboxylation. This
gives an estimated interaction energy of 5.6 kcal/mol that is 0.1
kcal/mol larger than the value of 5.5 kcal/mol calculated using
Figure 5. Dependence of observed second-order rate constant
7
−1 −1
the observed value of k /K = 1.1 × 10 M
s
(Table 3).
(
k /K ) for R235A mutant of ScOMPDC-catalyzed decarbox-
cat
m
cat
m obs
The intrinsic phosphodianion binding energy may be utilized
to induce electrostatic stress into the protein catalyst and/or
substrate, which is relieved at the transition state for the
ylation of truncated substrate FEO on concentration of added
phosphite dianion at pH 7.0, 25 °C, and I = 0.14 (NaCl). The solid
line shows the linear correlation of the experimental data to eq 3,
derived for Scheme 4, by using the values for the kinetic parameters
reported in Table 2.
43−45
enzyme-catalyzed decarboxylation reaction.
However, such
ground state destabilization would result in equal increases in
kcat and Km but should not affect the value of k /K . It is
cat
m
therefore not relevant to our analysis of the effect of the R235A
mutation on k /K for the ScOMPDC-catalyzed reactions of
cat
m
OMP and FOMP summarized in Table 3. The utilization of
F
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Biochemistry
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1
9,25,28,46,47
Table 3. Apparent Interaction Energies of Cationic Side
Chain of Arg235 with Rate-Determining Transition States
for Several ScOMPDC-Catalyzed Reactions
sis.
This is shown by Scheme 5 for ScOMPDC-
catalyzed reactions of OMP and h-FUMP, as the conversion of
a dominant inactive open form of OMPDC (E ) to the active
closed (E ) forms E ·OMP or E ·h-FUMP (K ≪ 1).
utilization of the dianion binding interactions to stabilize E
O
2
2,47
The
C
C
C
c
C
results in enzyme activation because of the increase in the
fraction of total OMPDC present as reactive E . The
substrate
(k /K )
m WT
interaction energy (kcal/mol)
C
cat
conformational change from E ·OMP to E ·OMP or from
O
C
(
k /K )
cat m R235A
E ·h-FUMP to E ·h-FUMP includes closure of a phospho-
O
C
a
1.2 × 104
b
OMP
5.5
dianion gripper loop (Pro202 to Val220) and of a hydrophobic
pyrimidine umbrella (Ala151−Thr165) over O-4 and C-5 of
the pyrimidine ring (Figure 6). The formation of a hydrogen
c
5
.6
a
FOMP
75
2.6
7.2
0
a
5
h-FUMP
2.1 × 10
d
EO
1
d
FEO
0.8
−0.1
5.8
3.1
HPO32 (1 M) + EO
−
e
1.9 × 10
4
HPO32 (1 M) + FEO
−
e
190
a
b
Calculated from data in Table 1. Observed stabilization of the rate-
c
determining transition state. Estimated stabilizing interaction if
chemistry were fully rate determining for ScOMPDC-catalyzed
d
decarboxylation of OMP. Calculated from data in Table 2.
e
Calculated from the effect of the R235A mutation on
(
k /K )
/(K ) (Table 2).
E·HPi d
cat
m
phosphodianion binding energy to induce electrostatic stress
between protein and substrate carboxylate anions, which is
relieved in the vinyl carbanion-like transition state for the
enzyme-catalyzed decarboxylation reaction, would result in a
smaller effect of the R235A mutation on the kinetic parameter
kcat for the enzyme-catalyzed deuterium exchange reaction of an
unstrained Michaelis complex to FUMP through a vinyl
carbanion-like transition state. We find, instead, that the R235A
Figure 6. Image that superimposes the partial X-ray crystal structure of
ScOMPDC complexed with BMP (PDB entry: 1DQX) over the
structure for unliganded ScOMPDC (PDB entry: 1DQW). The
movement of the phosphodianion gripper loop (Pro202 to Val220)
toward the hydrophobic pyrimidine umbrella (Ala151−Thr165) is
shown for the unliganded (orange) and liganded (olive green)
enzymes. The closure of these loops is proposed to be cooperative and
driven by the formation of a hydrogen bond between the side chains of
Gln215 from the gripper loop and of Ser 154 from the “umbrella”.
mutation results in a larger decrease in the k for the OMPDC-
ex
catalyzed deuterium exchange reaction of FUMP compared
with the kcat for enzyme-catalyzed decarboxylation of OMP.
The cationic side chain of Arg235 provides no stabilization of
the transition states for enzyme-catalyzed decarboxylation of
the truncated substrates EO and FEO but large 5.6 and 7.2
kcal/mol stabilization, respectively, of the transition states for
the decarboxylation and deuterium exchange reactions of
phosphorylated substrates OMP and FUMP (Table 3). The
absence of stabilizing interactions between this side chain and
the decarboxylation transition state, when there is no substrate
phosphodianion, shows that the large transition state
stabilization observed for the OMPDC-catalyzed reactions of
OMP and UMP is due entirely to stabilizing interactions
expressed at the enzyme−phosphodianion ion pair (Figure 1).
These interactions are not only expressed at the ground state
Michaelis complex (Km effect) because the R235A mutation
bond at EC between the side chains of Gln 215 from the
“gripper” loop and Ser154 from the “lid” shows that these
structural elements act cooperatively during the enzyme
48
conformational change. The mechanism for activation of
OMPDC for catalysis of decarboxylation and deuterium
exchange reactions, which we propose results from this
conformational change, has not been determined. A minimal
mechanism would include the following: (a) the utilization of
substrate binding energy for the optimal positioning of amino
4
9,50
acid residues at the enzyme active site;
(b) the utilization of
substrate binding energy to extrude solvent from the active site
(this will reduce the effective dielectric constant at the active
site cavity) to enhance stabilizing electrostatic interactions
4
6,49,50
results in decreases in kcat and k , respectively, for the
between the protein and transition state.
ex
ScOMPDC-catalyzed reactions of OMP and FUMP (Scheme
Effects on kcat and K . The ion pair between the side chain
m
5
).
cation of Arg235 and the phosphodianion of ligands (Figure 1)
appears to stabilize the enzyme−ligand complex, so that strong
phosphodianion binding interactions will favor tight substrate
binding. On the other hand, binding energy that is utilized to
drive an initially unfavorable, but activating, protein conforma-
tional change (Scheme 5) cannot be expressed as a decrease in
K and must instead result in an increase in k conversion of
We have proposed that the binding energy from the
protein−phosphodianion pair is utilized to drive an enzyme
conformational change that activates OMPDC for cataly-
Scheme 5
m
cat
25
the Michaelis complex to product.
Removing the side chain cation−phosphodianion pair by the
R235A mutation results in changes in both K or K for
m
d
formation of the Michaelis complex to OMP and h-FUMP,
respectively, and in kcat or k for conversion of the Michaelis
ex
G
dx.doi.org/10.1021/bi401117y | Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
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complex to product (Table 1). The 7.2 kcal/mol stabilizing
interaction between the side chain of Arg235 and the transition
state for the ScOMPDC-catalyzed deuterium exchange reaction
of h-FUMP (Table 3) reflects a 2.2 kcal/mol stabilization of the
Michaelis complex (Kd effect) and a larger 5.0 kcal/mol
decrease in the barrier for conversion of the Michaelis complex
to the reaction transition state (kex effect). By contrast, the
smaller 5.6 kcal/mol stabilizing interaction between the side
chain of Arg235 and the transition state for the decarboxylation
reaction reflects a large 4.0 kcal/mol stabilization of the
eq 6), and when unreactive E ·h-FUMP is the dominant
O
complex (K ≪ 1, Figure 7B), then changes in K will be
c
c
expressed as changes in the apparent reactivity of the Michaelis
complex ((k ) , eq 5), because the enzyme conformational
ex obs
change is a step on the pathway from the major complex E ·h-
O
FUMP to the reaction transition state. Mutations that result in
a change from K > 1 to K < 1 will be expressed as changes in
c
c
both (K ) and (k ) .
d obs
ex obs
OMP and FUMP bind to different protonated and neutral
42
13
forms of OMPDC with Kd = 1.0 μM and 20 μM,
respectively. The ligand binding is proposed to be controlled by
Michaelis complex to OMP (K effect) and only a 1.6 kcal/mol
m
13
decrease in the barrier to turnover of this complex (kcat effect).
the protonation state of the cationic side chain of Lys93.
Now, the effect of the R235A mutation is expressed mainly as a
change in (k ) for the ScOMPDC-catalyzed deuterium
k K
ex
c
[
h‐FUMP]
[h‐FUMP]
⎞
(
)
ex obs
1
+ Kc
v =
exchange reaction of h-FUMP. This is consistent with a small
favorable thermodynamic driving force for the enzyme
conformational change, from E ·h-FUMP to E ·h-FUMP for
K′
+ Kc
d
+
(1
)
(4)
(5)
(6)
O
C
wild-type ScOMPDC, and a large barrier to this conformational
change (K ≪ 1) for the R235A mutant enzyme. The higher
⎛
k K
ex
c
(
(
k ) = ⎜
⎟
c
ex obs
⎝
⎛
1 + K ⎠
c
affinity of OMP, compared with that of UMP, for OMPDC
reflects stabilizing binding interactions between the carboxylate
⎞
13
K′
d
side chain of OMP and the cationic side chain of Lys93. We
K ) = ⎜
⎟
d obs
1 + K ⎠
propose that these and possibly other interactions with this
⎝
c
−
−
CO2 are part of an extended network of cooperative
Figure 7 shows the complexes between h-FUMP and the
interactions that stabilize E ·OMP so that (K )
> (K )
.
C
c OMP
c FUMP
open form (E ) and the closed form (E ) of ScOMPDC, which
O
C
This more favorable conformational change at the Michaelis
complex to OMP compared with h-FUMP would result in the
larger observed fractional expression of the effect of the R235A
mutation on K (4.0/5.6 = 0.71) and a corresponding smaller
m
observed fractional expression on kcat (1.6/5.6 = 0.29) for the
enzyme-catalyzed decarboxylation reaction of OMP compared
with the fractional expression of the effect of the R235A
mutation on K (2.2/7.2 = 0.31) and on k (5.0/7.2 = 0.69) for
d
ex
the OMPDC-catalyzed deuterium exchange reaction of h-
FUMP. The difference in the effects of the R235A mutation on
the kinetic parameters for ScOMPDC-catalyzed decarboxyla-
tion of OMP and the deuterium exchange reaction of FUMP is
illustrated by the two free energy profiles shown in Figure 8,
and additional discussion for these differences is presented in
the figure legend.
Figure 7. Diagrams that illustrate the effect of a conformational change
that converts OMPDC from an inactive [nonproductive] open form to
an active [productive] closed form on the kinetic parameters (kex)obs
and (Kd)obs for the OMPDC-catalyzed deuterium exchange reaction of
h-FUMP to form d-FUMP (eqs 5 and 6).The steps leading to
formation of the major Michaelis complex are shown in red, and the
steps leading from this Michaelis complex to the transition state for
In summary, a mutation that destabilizes the closed form of
wild-type OMPDC by weakening the interactions with the
substrate phosphodianion will result in a decrease in substrate
affinity (Figure 7A, K effect) up to the point where K = 1 and
OMPDC-catalyzed deuterium exchange are shown in blue. (A) K ≫
c
1
. The major Michaelis complex is the productive complex E ·h-
C
FUMP and a change in K affects (K ) for substrate binding, but not
c
d
obs
d
c
(
k ) , for turnover to form product (eq 6). (B) K ≪ 1. The major
ex obs
c
(K ) ≈ K′ . A further destabilization of the closed enzyme
d obs
d
Michaelis complex is the nonproductive complex E ·h-FUMP, so that
the enzyme conformational change lies on the pathway from the major
O
will then be expressed as an increase in the barrier for
conversion of E ·h-FUMP to the rate-determining transition
O
complex E ·h-FUMP to the reaction transition state. A change in Kc
O
state (Figure 7B, kex effect). The partitioning of the overall
effect on k /K or k /K into effects on substrate binding and
now results mainly in a change in (kex)obs for turnover of h-FUMP (eq
cat
m
ex
d
5).
catalysis depends upon the initial value of Kc for the
conformational change at the Michaelis complex, and we
propose that this is more favorable for the binding and reaction
of OMP compared with h-FUMP.
Stabilization of Vinyl Carbanion-Like Transition
States. Binding interactions between the phosphodianion
and the side chain cation of Arg235 provide a strong
stabilization of the vinyl carbanion-like transition states for
both enzyme-catalyzed decarboxylation and deuterium ex-
change reactions (Table 1). Similarly, interactions between
the phosphodianion and exogenous guanidinium cation provide
similar activation of R235A mutant enzyme-catalyzed decar-
boxylation and deuterium exchange reactions (Figure 4). This
result is consistent with a similar stabilization of these two vinyl
are interconverted with an equilibrium constant K . Equations
c
4
−6 define the relationships between K , K′ , and k from
c
d
ex
Figure 7 and the observed kinetic parameters (k ) and
ex obs
(
K )
for the ScOMPDC-catalyzed deuterium exchange
d obs
reactions of h-FUMP. A similar set of equations for (k )
cat obs
and (K )
can be written for the ScOMPDC-catalyzed
m obs
decarboxylation of OMP. Figure 7 shows limiting cases for
the effect of a change in the protein cation−phosphodianion
interaction, which affects the value of K , on the observed
c
kinetic parameters. When E ·h-FUMP is the dominant
C
complex (K ≫ 1, Figure 7A), then changes in K will be
c
c
expressed as changes in the observed substrate affinity ((K ) ,
d obs
H
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Biochemistry
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Figure 8. Free energy reaction profiles that illustrate different effects of R235A mutation on kinetic parameters. Profiles shown are for ScOMPDC-
catalyzed decarboxylation of OMP to form UMP (profile on left) and the deuterium exchange reaction of h-FUMP to form d-FUMP (profile on
right). The binding energy of R235A and other residues in the phosphodianion gripper loop is utilized to drive the conversion of open enzyme E to
O
closed enzyme E . This binding energy is partly expressed at the Michaelis complex when loop closure at the Michaelis complex is
C
thermodynamically favorable (K > 1, eqs 4−6). The substrate binding energy (ΔG ) of OMP is greater than that for FUMP, and we propose that
c
S wt
−
this reflects the utilization of the binding interactions with the −CO group of OMP to drive the conversion of E to E (ΔG′ ). The overall effect
2
O
C
c
‡
of the R235A mutation on the barrier for the deuterium exchange (ΔΔG
= 7.2 kcal/mol) is larger than that for the decarboxylation reaction
UMP
‡
(ΔΔG
= 5.6 kcal/mol). The R235A mutation results mainly in a decrease in ΔG for the decarboxylation reaction (ΔΔG = 4 kcal/mol). We
OMP
S
S
propose that the Michaelis complex between OMP and the R235A mutant enzyme exists mainly in the nonproductive open form ([E ·OMP]
),
O
R235A
because there is insufficient binding energy at this Michaelis complex enzyme to drive the enzyme conformational change (K < 1, eqs 4−6). The
c
‡
effect of the R235A mutation on kcat (ΔΔG = 1.6 kcal/mol) is shown as the 1.6 kcal/mol barrier for conversion of [E ·OMP]R235A to the
k
cat
O
productive complex ([E ·OMP]R235A). The smaller binding energy from FUMP compared with OMP available to drive the enzyme conformational
C
change ((Kc)FUMP < (Kc)OMP, eqs 4−6) results in the smaller the effect of the R235A mutation on ΔG (ΔΔG = 2.2 kcal/mol) and a large 5
S
S
kcal/mol barrier to conversion of nonproductive [E ·FUMP]
to productive ([E ·FUMP]
that is added to the barrier for conversion of
O
R235A
C
R235A
‡
[
EO·FUMP]R235A to product (ΔΔG k = 5 kcal/mol).
cat
carbanion-like transition states by interaction with Gua+.13,14,20
This limited set of data shows that the deuterium exchange and
decarboxylation reactions catalyzed by this mutant OMPDC are
entropic cost to formation of a network of hydrogen bonding
and ionic interactions with the substrate phosphodianion.
51,52
We propose that there is a related greater cooperativity for
FUMP compared with OMP in the expression of the
phosphodianion binding interactions with the amino acid side
chains of Gln215, Tyr217, and Arg235 (Figure 1) and that the
large 7.2 kcal/mol effect of the R235A mutation on the stability
of the transition state for the deuterium exchange reaction of h-
FUMP reflects not only the loss of the interactions with the
excised side chain (≈5.6 kcal/mol determined for the reaction
of OMP) but also a ca. 1.6 kcal/mol weakening of interactions
between the phosphodianion and the side chains of the
+
activated to a similar degree by Gua , and that the side chain
27
shows similar effective molarities in the two reactions.
The apparent interaction of the cationic side chain of Arg235
with the transition state for the ScOMPDC-catalyzed deuterium
exchange reaction (7.2 kcal/mol) is larger than the interaction
with the transition state for the enzyme-catalyzed decarbox-
ylation reaction (5.6 kcal/mol), but the 5.6 kcal/mol
interaction energy is 50% of the total 11.2 kcal/mol
contribution of the phosphodianion binding energy to the
18
stabilization of the decarboxylation transition state, whereas
the 7.2 kcal/mol interaction is a larger 78% of the total 9.2
kcal/mol contribution of the phosphodianion binding energy to
53
remaining gripper residues Gln215 and Tyr217 (Figure 1).
Once again, we propose that the phosphodianion binding
interactions are more fully maintained at the transition state for
the R235A mutant ScOMPDC-catalyzed decarboxylation,
compared with the deuterium exchange reaction, because the
interactions between OMPDC and the pyrimidine carboxylate
have the effect of reducing the entropy of the bound substrate
20
the stabilization of the exchange transition state.
We speculate that the 2 kcal/mol larger phosphodianion
binding energy utilized to stabilize the transition state for
OMPDC-catalyzed decarboxylation of OMP compared with
the deuterium exchange reaction of h-FUMP reflects the tighter
fit of OMP at the enzyme active site. This minimizes the
52
by “locking” the ligand into a rigid reactive conformation.
I
dx.doi.org/10.1021/bi401117y | Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
ScOMPDC-Catalyzed Decarboxylation of FOMP and
FEO. The cationic side chain of Arg235 provides similar 5.6 and
of FUMP compared with the OMPDC-catalyzed decarbox-
ylation reaction.
There are interesting differences in the mechanisms for the
utilization of dianion binding interactions in the activation of
triosephosphate isomerase for catalysis of the truncated
substrate glycolaldehyde (GA, Scheme 6) and of OMPDC
52
5.8 kcal/mol stabilization of the transition states for the
ScOMPDC-catalyzed reactions of OMP and the substrate
2−
pieces EO + HPO3 , respectively, and smaller but likewise
similar, 2.6 and 3.1 kcal/mol stabilization of the transition states
for the enzyme-catalyzed reactions of FOMP and the substrate
2−
pieces FEO + HPO3 (Table 3). The similar interaction of this
side chain with the transition states for the reactions of the
whole substrates OMP or FOMP and the corresponding
Scheme 6
2
−
2−
substrate pieces EO + HPO3 or FEO + HPO3 is consistent
with the conclusion that the transition states for reactions of
related whole substrates and substrate pieces show essentially
18−20,48
the same interaction with the protein catalyst.
The main
effect of this covalent attachment is to reduce the entropic
barrier for the unimolecular reaction of the whole substrate,
compared with the bimolecular reaction of the corresponding
for catalysis of the decarboxylation and deuterium exchange
1
8,20,22
reactions of EO and FEU, respectively (Scheme 2).
The
2
3
−
5
1,54
binding loci for the small carbon acid GA and for HPO are
pieces.
The difference in the stabilization of the transition states for
55,56
overlapping.
The sites behave independently to the extent
2
−
that some structural mutations, which have little effect on
enzyme activation by phosphite dianion, result in a decrease in
the enzyme reactivity toward deprotonation of glycolalde-
ScOMPDC-catalyzed reactions of FOMP or FEO + HPO
3
2−
compared with OMP or EO + HPO3 by interaction with the
cationic side chain of Arg235 reflects the different rate
determining steps for the wild-type enzyme-catalyzed reactions
5
7
hyde. Binding of dianion activators to TIM triggers
movement of active site loops 6 and loop 7. This places the
active site base from Glu167 in a reactive conformation and
2
8,29,34
of the two types of substrates.
The values of the kinetic
parameters k /K (Table 1) or [(k /K ) ]/K ] (Table 2)
cat
m
cat
m E·Pi
d
55,56
swings the side chain of Ile172 toward the catalytic base,
for the wild-type ScOMPDC-catalyzed reactions of OMP or
2
−
with the effect of shielding the substrate and the catalytic
EO + HPO3 , respectively, are limited by the rate of the
chemical decarboxylation step, whereas values for the wild-type
55
carboxylate from interaction with bulk solvent. The results of
2−
studies on the effect of the I172A mutation on the kinetic
enzyme-catalyzed reactions of FOMP or FEO + HPO3 are
limited by rate constant for formation of the Michaelis complex
2−
parameters for the TIM-catalyzed reactions of GA and HPO3
2
8,29,34
provide strong evidence for the conclusion that interactions
between the hydrophobic side chain of I172 and the catalytic
carboxylate from Glu167 activate TIM for catalysis of
deprotonation of carbon by enhancing the basicity of this
to substrate.
The effect of the loss of the side chain
interaction at the R235A mutant enzyme is essentially entirely
expressed as the change in the values of the kinetic parameters
for the wild-type ScOMPDC-catalyzed reactions of substrates
58−60
carboxylate ion.
2−
OMP or EO + HPO3 , for which the chemical step is rate
determining. By comparison, the effect of the R235A mutation
should not result in a decrease in k /K or [(k /K ) ]/Kd]
By comparison to TIM, the binding loci for EO and HPO32−
are well separated at the active site of OMPDC. Binding of the
dianion activator also triggers large movement of a
phosphodianion gripper loop, but this loop appears too remote
from the pyrimidine binding site to result in a large activation of
OMPDC for catalysis of decarboxylation of EO (Figures 1 and
cat
m
cat
m E·Pi
2−
for the reactions of FOMP or FEO + HPO3 , until there is a
change in the rate-determining step. Our results show that this
change in rate determining step only occurs after there is a ca. 3
kcal/mol increase in the barrier to the decarboxylation of
6
). The results reported in this paper provide support for the
2
3
enzyme-bound FOMP or FEO + HPO .
conclusion that binding of the dianion activator triggers a much
more extensive cooperative conformational change, which
includes not only movement of the phosphodianion gripper
loop but also the enzyme-activating closure of the pyrimidine
umbrella. The dense network of hydrogen bonds that involve
Ser154, from the pyrimidine umbrella, and Gln215, from the
gripper loop should promote cooperative closure of these
Summary. We have rationalized these data by a model in
which the following apply: (1) The Michaelis complex between
FUMP and R235A mutant OMPDC exists mainly in the open
conformation (Figure 7B), so that the interactions with the
cationic side chain of R235 at wild-type OMPDC result mainly
in an increase in kex for the deuterium exchange reaction
48
(
Figure 8). (2) The stabilizing interactions between OMPDC
structural elements.
−
and the −CO2 group of OMP result in a shift in this
conformational equilibrium to the closed form of OMPDC, so
that the interactions with the cationic side chain of R235 at
ASSOCIATED CONTENT
Supporting Information
■
*
S
wild-type OMPDC now result mainly in a decrease in K for
Table S1: UV wavelengths monitored in determining initial
velocity data reported in Figure 2 for R235A ScOMPDC-
catalyzed decarboxylation of OMP at different ionic strengths.
This material is available free of charge via the Internet at
http://pubs.acs.org.
m
decarboxylation (Figure 8). (3) The stabilizing interactions
−
between OMPDC and the −CO group of OMP are utilized
2
to immobilize the substrate in ScOMPDC: the removal of the
−
−
CO2 group results in a greater conformational flexibility of
enzyme bound FUMP, a larger requirement for the utilization
of the phosphodianion binding interactions to immobilize this
substrate with the result that a smaller fraction of the total
dianion binding energy is available to stabilize the transition
state for the ScOMPDC-catalyzed deuterium exchange reaction
AUTHOR INFORMATION
Corresponding Author
*J. P. Richard. Tel: (716) 645-4232. Fax: (716) 645-6963. E-
mail: jrichard@buffalo.edu.
■
J
dx.doi.org/10.1021/bi401117y | Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Funding
(13) Tsang, W.-Y., Wood, B. M., Wong, F. M., Wu, W., Gerlt, J. A.,
Amyes, T. L., and Richard, J. P. (2012) Proton Transfer from C-6 of
Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophos-
phate Decarboxylase: Formation and Stability of a Vinyl Carbanion
Intermediate and the Effect of a 5-Fluoro Substituent. J. Am. Chem.
Soc. 134, 14580−14594.
This work was supported by Grants GM 039754 to J.P.R. and
GM 065155 to J.A.G. from the National Institutes of Health.
Notes
The authors declare no competing financial interest.
(14) Amyes, T. L., Wood, B. M., Chan, K., Gerlt, J. A., and Richard, J.
P. (2008) Formation and Stability of a Vinyl Carbanion at the Active
ABBREVIATIONS
■
Site of Orotidine 5′-Monophosphate Decarboxylase: pK of the C-6
a
ScOMPDC, orotidine 5′-monophosphate decarboxylase from S.
cerevisiae; PRPP synthase, phosphoribosylpyrophosphate syn-
thase; ORTase, orotate phosphoribosyl transferase; TIM,
triosephosphate isomerase; OMP, orotidine 5′-monophos-
phate; UMP, uridine 5′-monophosphate; FOMP, 5-fluoroor-
otidine 5′-monophosphate; FUMP, 5-fluorouridine 5′-mono-
phosphate; h-FUMP, FUMP labeled with hydrogen at C-6; d-
FUMP, FUMP labeled with deuterium at C-6; BMP, 6-
hydroxyuridine 5′-monophosphate; EO, 1-(β-D -
erythrofuranosyl)orotic acid; EU, 1-(β-D-erythrofuranosyl)-
uracil; FEO, 1-(β-D-erythrofuranosyl)-5-fluoroorotic acid;
Proton of Enzyme-Bound UMP. J. Am. Chem. Soc. 130, 1574−1575.
(15) Chan, K. K., Wood, B. M., Fedorov, A. A., Fedorov, E. V., Imker,
H. J., Amyes, T. L., Richard, J. P., Almo, S. C., and Gerlt, J. A. (2009)
Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-
Catalyzed Reaction: Evidence for Substrate Destabilization. Biochem-
istry 48, 5518−5531.
(16) Toth, K., Amyes, T. L., Wood, B. M., Chan, K., Gerlt, J. A., and
Richard, J. P. (2010) Product Deuterium Isotope Effects for Orotidine
5
′-Monophosphate Decarboxylase: Effect of Changing Substrate and
Enzyme Structure on the Partitioning of the Vinyl Carbanion Reaction
Intermediate. J. Am. Chem. Soc. 132, 7018−7024.
(17) Toth, K., Amyes, T. L., Wood, B. M., Chan, K., Gerlt, J. A., and
+
FEU, 1-(β-D-erythrofuranosyl)-5-fluorouracil; Gua , guanidi-
nium cation; GA, glycolaldehyde; MOPS, 3-(N-morpholino)-
propanesulfonic acid; NMR, nuclear magnetic resonance
Richard, J. P. (2007) Product Deuterium Isotope Effect for Orotidine
′-Monophosphate Decarboxylase: Evidence for the Existence of a
Short-Lived Carbanion Intermediate. J. Am. Chem. Soc. 129, 12946−
2947.
18) Amyes, T. L., Richard, J. P., and Tait, J. J. (2005) Activation of
5
1
(
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