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

Article abstract of DOI:10.1021/ja102408k

A product deuterium isotope effect (PIE) of 1.0 was determined as the ratio of the yields of [6-¹H]-uridine 5′-monophosphate (50%) and [6-²H]-uridine 5′-monophosphate (50%) from the decarboxylation of orotidine 5′-monophosphate (OMP) in 50/50 (v/v) HOH/DOD catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae, Methanothermobacter thermautotrophicus, and Escherichia coli. This unitary PIE eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO₂ in a concerted reaction. We propose that the complete lack of selectivity for the reaction of solvent H and D, which is implied by the value of PIE = 1.0, is enforced by restricted C-N bond rotation of the -CH₂-NL₃⁺ group of the side chain of Lys-93. A smaller PIE of 0.93 was determined as the ratio of the product yields for OMPDC-catalyzed decarboxylation of 5-fluoroorotidine 5′-monophosphate (5-FOMP) in 50/50 (v/v) HOH/DOD. Mutations on the following important active-site residues of OMPDC from S. cerevisiae have no effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A, Y217A, Q215A, S124A, and S154A/Q215A.

Full text of DOI:10.1021/ja102408k

Published on Web 05/04/2010
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
†
†
#
#
#
Krisztina Toth, Tina L. Amyes, Bryant M. Wood, Kui Chan, John A. Gerlt, and
,
†
John P. Richard*
Department of Chemistry, UniVersity at Buffalo, Buffalo, New York 14260, and Departments of
Biochemistry and Chemistry, UniVersity of Illinois, Urbana, Illinois 61801
Received March 22, 2010; E-mail: jrichard@buffalo.edu
Abstract: A product deuterium isotope effect (PIE) of 1.0 was determined as the ratio of the yields of
1
2
[
6- H]-uridine 5′-monophosphate (50%) and [6- H]-uridine 5′-monophosphate (50%) from the decarboxylation
of orotidine 5′-monophosphate (OMP) in 50/50 (v/v) HOH/DOD catalyzed by orotidine 5′-monophosphate
decarboxylase (OMPDC) from Saccharomyces cerevisiae, Methanothermobacter thermautotrophicus, and
Escherichia coli. This unitary PIE eliminates a proposed mechanism for enzyme-catalyzed decarboxylation
2
in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO in a
concerted reaction. We propose that the complete lack of selectivity for the reaction of solvent H and D,
+
which is implied by the value of PIE ) 1.0, is enforced by restricted C-N bond rotation of the -CH
2
-NL
3
group of the side chain of Lys-93. A smaller PIE of 0.93 was determined as the ratio of the product yields
for OMPDC-catalyzed decarboxylation of 5-fluoroorotidine 5′-monophosphate (5-FOMP) in 50/50 (v/v) HOH/
DOD. Mutations on the following important active-site residues of OMPDC from S. cerevisiae have no
effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A,
Y217A, Q215A, S124A, and S154A/Q215A.
Introduction
OMPDC is assayed in the direction of decarboxylation of
OMP, and mechanistic studies have naturally focused on the
loss of CO from substrate, which is the first stage in a stepwise
Orotidine 5′-monophosphate decarboxylase (OMPDC) is a
remarkable enzyme because it employs no metal ions or other
cofactors, yet it effects an enormous ca. 30 kcal/mol stabilization
of the transition state for the decarboxylation of orotidine 5′-
monophosphate (OMP) to give uridine 5′-monophosphate
2
reaction mechanism. The second stage of proton transfer
between the enzyme and substrate has received much less
2
,9
consideration in experimental and theoretical studies. This
strong focus on the first reaction stage has resulted in a
significant, but unappreciated, gap in our understanding of the
enzymatic reaction mechanism, which can only be fully defined
by studies that encompass the whole reaction.
1
-3
(
UMP, Scheme 1).
A significant fraction of this rate
acceleration is obtained from utilization of the intrinsic phos-
phate binding energy in stabilization of the decarboxylation
4
,5
transition state. The electrophilic substitution of -CO
proton may proceed by two reaction steps or stages: loss of
CO to form the putative vinyl carbanion reaction intermediate
2
by a
2
Loss of CO and the addition of a proton to carbon-6 of OMP
may proceed by the fully stepwise reaction mechanism shown
in Scheme 1, or by a concerted mechanism that avoids the
formation of the vinyl carbanion reaction intermediate. The latter
was suggested for OMPDC-catalyzed decarboxylation, in order
to provide a mechanism for avoiding formation of the unstable
vinyl carbanion intermediate, while at the same time providing
2
and protonation of this carbanion by an acidic catalytic residue
to form UMP. It has also been suggested that these two steps
might be coupled in a concerted electrophilic substitution
6
-8
reaction.
for transition-state stabilization from electrophilic push by the
†
University at Buffalo.
University of Illinois.
6-8
#
transferred proton.
This distinction between stepwise and
(
1) Callahan, B. P.; Miller, B. G. Bioorg. Chem. 2007, 35, 465–469.
concerted reaction mechanisms for proton transfer from enzyme
to substrate can only be made by a direct study of the OMPDC-
catalyzed proton-transfer reaction.
The product deuterium isotope effect (PIE, Scheme 2),
determined directly as the yield of products of reaction of -H
(
2) Miller, B. G.; Wolfenden, R. Annu. ReV. Biochem. 2002, 71, 847–
8
85.
(
(
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(
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15.
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2
7
018 9 J. AM. CHEM. SOC. 2010, 132, 7018–7024
10.1021/ja102408k  2010 American Chemical Society

Product Deuterium Isotope Effects on Enzyme-Catalyzed Decarboxylation
A R T I C L E S
Scheme 1
Scheme 2
results provide strong evidence that the step that determines
the yields of H- and D-labeled products is decarboxylation of
enzyme-bound substrate, and that protonation of the carbanion
intermediate of decarboxylation is faster than steps that would
exchange acidic hydrons and allow the intermediate to select
between reaction with -H and -D. The observed PIE shows a
remarkable insensitivity to changes in substrate reactivity and
enzyme catalytic activity.
and of -D in 50/50 HOH/DOD, provides a measure of the
relative electrophilic push of the transferred -H and -D at the
transition state for chemical and enzymatic reactions. We have
determined PIEs as large as 8 for thermoneutral proton transfer
from carboxylic acids to ring-substituted R-methoxystyrenes,
where there is apparently the full loss of zero-point energy at
the transferred hydron at the rate-determining transition state.
The PIE decreases below this maximum value for either
thermodynamically favorable or unfavorable proton-transfer
Experimental Section
Materials. Orotidine 5′-monophosphate trisodium salt (99%) was
purchased from Sigma or prepared by chemical methods from
uridine 5′-monophosphate using modifications of literature pro-
2
0-22
1
0,11
cedures.
3-(N-Morpholino)propanesulfonic acid (MOPS,
g99.5%) was purchased from Fluka. Water was from a Milli-Q
purification system. Deuterium-labeled water (99.9% D), DCl (35
wt %, 99.9% D), and KOD (40 wt %, >98% D) were from
Cambridge Isotope Laboratories. 5-FOMP was prepared by orotate
phosphoribosyl transferase-catalyzed coupling of 5-fluoroorotate and
5′-phosphoribosyl-1′-pyrophosphate by following a literature pro-
1
1-13
reactions.
PIEs of 1.0 for nonenzymatic reactions in
14-18
aqueous solution are almost never observed
but are possible
for a complex reaction mechanism where proton transfer occurs
after the step that determines the yields of hydrogen- and
deuterium-labeled reaction products. In this case the deuterium
enrichment of product will be determined by the initial
deuterium enrichment of the catalytic acid.
23
cedure. All other chemicals were reagent grade or better and were
used without further purification.
The S155C mutant of OMPDC from Saccharomyces cereVisiae
2
3,24
(
ScOMPDC) was prepared as described in earlier work.
This
enzyme was used as a model for the wild-type enzyme, because
the mutant is more stable than but kinetically and structurally
essentially identical with wild-type ScOMPDC. OMPDCs from
Methanothermobacter thermautotrophicus (MtOMPDC) and from
We recently reported a fast and convenient method to
1
determine the yields of [6- H]-uridine 5′-monophosphate ([6-
1
2
2
H]-UMP) and [6- H]-uridine 5′-monophosphate ([6- H]-UMP)
from the decarboxylation of OMP in 50/50 (v/v) HOH/DOD
catalyzed by OMPDC. The ratio of the yields of these two
products was determined to be equal to the isotopic enrichment
Escherichia coli (EcOMPDC) were also prepared and purified as
23,25
described in earlier work.
The gene for the S155C mutant of
ScOMPDC was used as the starting point for preparation of the
1
9
24
24
24
26
of solvent, so that the PIE on this reaction is 1.0. We now
report (a) the full experimental details from our earlier prelimi-
nary communication and a significant modification of the
S154A, Q215A, S154A/Q215A, and R235A mutant en-
zymes. Similar procedures were followed in preparing the Y217A
mutant enzyme of ScOMPDC (see Supporting Information).
1
1
NMR Analyses. H NMR spectra (ca. 30 transients) were
protocol for H NMR analysis of the deuterium enrichment of
recorded on a Varian Unity Inova-500 spectrometer using a sweep
product UMP; (b) the PIE on OMPDC-catalyzed decarboxyla-
tion of 5-FOMP, a substrate that is activated for decarboxylation
by the C-5 fluorine; and (c) PIEs on decarboxylation of OMP
and 5-FOMP by several site-directed mutants of OMPDC. These
width of 6000 Hz, a 90° pulse angle, an acquisition time of 6 s,
1
and a relaxation delay between pulses of 120 s (>7T ), with
suppression of the water peak. Baselines were subjected to first-
order drift corrections before integration of the signals. Chemical
1
9
shifts are reported relative to HOD at 4.67 ppm. F NMR spectra
(64 transients) were recorded on a Varian Unity Inova-500
spectrometer using a spectral width of 50 000 Hz, a 90° pulse angle,
(
(
(
10) Tsang, W.-Y.; Richard, J. P. J. Am. Chem. Soc. 2007, 129, 10330–
0331.
11) Tsang, W.-Y.; Richard, J. P. J. Am. Chem. Soc. 2009, 131, 13952–
1
1
3962.
12) Bell, R. P. The Proton in Chemistry, 2nd ed.; Cornell University Press:
Ithaca, NY, 1973.
(20) Smiley, J. A.; Paneth, P.; O’Leary, M. H.; Bell, J. B.; Jones, M. E.
Biochemistry 1991, 30, 6216–6223.
(
(
(
13) Westheimer, F. H. Chem. ReV. 1961, 61, 265–273.
14) Corfield, J. R.; Trippett, S. Chem. Commun. 1970, 1267.
15) Alexander, R.; Asomaning, W. A.; Eaborn, C.; Jenkins, I. D.; Walton,
D. R. M. J. Chem. Soc., Perkin Trans. 2 1974, 490–494.
16) Eaborn, C.; Walton, D. R. M.; Seconi, G. J. Chem. Soc., Perkin Trans.
(21) Van Vleet, J. L.; Reinhardt, L. A.; Miller, B. G.; Sievers, A.; Cleland,
W. W. Biochemistry 2008, 47, 798–803.
(22) Miller, B. G.; Butterfoss, G. L.; Short, S. A.; Wolfenden, R.
Biochemistry 2001, 40, 6227–6232.
(
(
(
(
(23) Wood, B. M.; Chan, K. K.; Amyes, T. L.; Richard, J. P.; Gerlt, J. A.
Biochemistry 2009, 48, 5510–5517.
2
1976, 1857–1861.
17) Macciantelli, D.; Seconi, G.; Eaborn, C. J. Chem. Soc., Perkin Trans.
1978, 834–838.
18) Fishbein, J. C.; Jencks, W. P. J. Am. Chem. Soc. 1988, 110, 5087–
(24) Barnett, S. A.; Amyes, T. L.; Wood, B. M.; Gerlt, J. A.; Richard,
J. P. Biochemistry 2008, 47, 7785–7787.
2
(25) Toth, K.; Amyes, T. L.; Wood, B. M.; Chan, K. K.; Gerlt, J. A.;
Richard, J. P. Biochemistry 2009, 48, 8006–8013.
5
095.
19) Toth, K.; Amyes, T. L.; Wood, B. M.; Chan, K.; Gerlt, J. A.; Richard,
(26) Barnett, S. A.; Amyes, T. L.; McKay Wood, B.; Gerlt, J. A.; Richard,
J. P. Biochemistry 2010, 49, 824–826.
J. P. J. Am. Chem. Soc. 2007, 129, 12946–12947.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 20, 2010 7019

A R T I C L E S
Toth et al.
Scheme 3
a 2.6 s acquisition time, and a relaxation delay between pulses of
effect of 1.0 (Table 1) was determined as the ratio of the
1
2
0 s (>7T
1
). The receiver gating time was optimized to obtain a
integrated area A of the doublet due to the C-6 proton of [6- H]-
H
flat baseline. Baselines were subjected to first-order drift correction
before integration of the signals. Chemical shifts are reported
relative to a neat trifluoracetic acid external standard for which
D
UMP at 7.990 ppm and A of the collapsed singlet due to the
2
19
C-5 proton of [6- H]-UMP at 5.865 ppm (eq 1).
2
7
δ ) -78.5 ppm.
[
[
P ]
AH
H
OMPDC-Catalyzed Decarboxylation. Stock solutions of OMP
and 5-FOMP were prepared in 50/50 HOH/DOD, and their
concentrations were determined from the absorbance in 0.1 M HCl
PIE )
)
(1)
P
]
D
A
D
-1
-1
at 267 nm using ꢀ ) 9400 and 10 200 M cm for OMP and
The spectrum of UMP at pL 9.7 shows a better resolution of
the signals for the C-5 pyrimidine and the C-1′ anomeric
hydrogen, because of the effect of partial ionization of N-3 of
5
-FOMP, respectively. The concentration of wild-type ScOMPDC,
EcOMPDC, and MtOMPDC in stock solutions was determined
3
0
from the absorbance at 280 nm using values of ꢀ ) 29 900, 10 100,
UMP (pK ) 9.45) on chemical shift. Figure 1B shows a
a
and 6100 M- cm , respectively, calculated using the ProtParam
1
-1 25
1
representative partial H NMR spectrum, at pL 9.7, of the UMP
product of decarboxylation of OMP (2 mM) catalyzed by the
R235A mutant of ScOMPDC (13 µM) in 50/50 (v/v) HOH/
2
8,29
tool available on the ExPASy server.
The concentration of
mutant OMPDCs in stock solutions was determined from the
absorbance at 280 nm using ꢀ ) 29 900 or 28 420 M- cm for
the Y217A mutant, calculated using the ProtParam tool. All enzyme
assays were carried out at 25 °C in 10 mM MOPS (50% free base)
at pH 7.1 and I ) 0.105 (NaCl). Standard assays described in earlier
work were used to determine the activity of OMPDC for decar-
1
-1
2
4
boxylation of OMP. Decarboxylation of OMP (2 mM) catalyzed
by wild-type and mutant OMPDCs at 25 °C was carried out in
50/50 HOH/DOD (2 mL) that contained 50 mM MOPS buffer (50%
free base) at pL 7.4 with ionic strength of 0.1 (NaCl). The reactions
were initiated by addition of OMPDC (0.01-30 µM). The reaction
was allowed to proceed until ca. 95% complete, at which time (x
min) an 800 µL aliquot was withdrawn and the enzyme was
removed by ultrafiltration using Microcon centrifugal filter units
(
8
10K MWCO). After an additional reaction time of x min, a second
00 µL aliquot was withdrawn and treated in the same manner.
The filtered samples were frozen until NMR analysis of the
deuterium enrichment of the 6-H position. Prior to 1H NMR
analyses, the solution pL was adjusted to 9.5-10 using NaOD, in
order to increase the resolution of the signals for the 5-H and 1′-H
of UMP. The same procedures were followed in studies of OMPDC-
catalyzed decarboxylation of 5-FOMP (2 mM), except that the pL
was kept at the initial value of 7.4 for 19F NMR analyses.
Results
The yields of H- and D-labeled products from decarboxylation
of OMP catalyzed by OMPDC in 50/50 (v/v) HOH/DOD
1
(
Scheme 3, X ) H) were determined by H NMR spectroscopy
1
at 500 MHz. Figure 1A shows a representative partial H NMR
spectrum at pL 7.4 of UMP obtained from the decarboxylation
of OMP (2 mM) catalyzed by ScOMPDC (C155S mutant, 0.024
µM, 200 min reaction time) in 50/50 (v/v) HOH/DOD at pL
19
1
7
.4 and 25 °C (I ) 0.10, NaCl). The product deuterium isotope
Figure 1. (A) Partial H NMR spectrum (500 MHz) at pL 7.4 of UMP
from decarboxylation of OMP (2 mM) catalyzed by ScOMPDC (0.024 µM)
in 50/50 (v/v) HOH/DOD at 25 °C. Key: (9) doublet due to the C-6 proton
(
27) Silverstein, R. M.; Webster, F. X.; Kiemble, D. J. Spectrophotometric
Identification of Organic Compounds, 7th ed.; John Wiley & Sons
Inc.: New York, 2005; p 323.
1
of [6- H]-UMP; (b) double doublets (poorly resolved) due to the anomeric
1
2
protons of [6- H]-UMP and [6- H]-UMP; ([) doublet due to the C-5 proton
1
2
of [6- H]-UMP; and (1) collapsed singlet due to the C-5 proton of [6- H]-
UMP. (B) Partial H NMR spectrum at pL 9.7 of [6- H]-UMP and [6- H]-
UMP. Key: (b) double doublets due to the anomeric protons of [6- H]-UMP
and [6- H]-UMP; ([) doublet due to the C-5 proton of [6- H]-UMP; and (1)
(
28) Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R. D.;
Bairoch, A. Nucleic Acids Res. 2003, 31, 3784–3788.
1
1
2
1
(
29) Protein Identification and Analysis Tools on the ExPASy SerVer;
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R.,
Appel, R. D., Bairoch, A., Eds.; Humana Press Inc.: Totowa, NJ, 2005.
2
1
2
collapsed singlet due to the C-5 proton of [6- H]-UMP.
7
020 J. AM. CHEM. SOC. 9 VOL. 132, NO. 20, 2010

Product Deuterium Isotope Effects on Enzyme-Catalyzed Decarboxylation
A R T I C L E S
Table 1. Product Deuterium Isotope Effects for Catalysis of Decarboxylation of OMP and 5-FOMP by Wild-Type and Mutants of Orotidine
a
5
-Monophosphate Decarboxylase
-1
-1
b
c
enzyme
kcat/K
m
(M
s
)
substrate
[E]/µM
reaction time (min)
PIE
5
6
7
7
d
d
E. coli
wild-type
6.4 × 10
3.3 × 10
1.1 × 10
OMP
0.044
200, 400
1.05 ( 0.04
1.01 ( 0.07
M. thermautotrophicus
S. cereVisiae
wild-type
wild-type
OMP
OMP
0.042
600, 1200
d
0.024
200
17, 33
8, 16
1.0
e
f
0
.47
1.02 ( 0.01
0.93 ( 0.02
1.03 ( 0.01
0.92 ( 0.04
1.06 ( 0.01
0.95 ( 0.01
1.02 ( 0.02
0.95 ( 0.02
1.04 ( 0.01
0.92 ( 0.01
1.03 ( 0.02
0.93 ( 0.02
1
610
.2 × 10
5-FOMP
OMP
5-FOMP
OMP
5-FOMP
OMP
5-FOMP
OMP
0.13
13
0.054
0.026
0.010
28
1.6
12
0.38
0.59
0.40
e
f
R235A
15, 30
15, 30
200, 400
40, 80
55, 110
5, 10
240, 480
150, 300
50, 100
45, 90
5
5
6
1
.3 × 10
e
f
Q215A
2.5 × 10
2
630
.6 × 10
e
f
S154A
5
4
4
2
380
.7 × 10
e
f
S154A/Q215A
Y217A
7
2500
.7 × 10
5-FOMP
OMP
5-FOMP
e
f
1
.3 × 10
a
b
For reactions in 50/50 (v/v) HOH/DOD at pL 7.4 and 25 °C (I ) 0.10, NaCl). The product yields were first determined after ca. 95% completion
c
of the decarboxylation reaction (see text). The ratio of the yields of the H- and D-labeled products for reactions in 50/50 (v/v) HOH/DOD. In all cases
but one, the reported PIEs are the average of the values determined at two different reaction times. The quoted uncertainty gives the range of these two
d
1
1
values. The ratio of the area from H NMR of the doublet for the C-6 hydrogen of [6- H]-UMP and of the collapsed singlet for the C-5 hydrogen of
2
e
1
[
6- H]-UMP determined from spectral data at pL 7.4 (Figure 1A). The ratio of the area from H NMR of the doublet for the C-5 hydrogen
1
2
f
of [6- H]-UMP and of the collapsed singlet for the C-5 hydrogen of [6- H]-UMP determined from spectral data at pL 9.5-10 (Figure 1B). The ratio of
the area from 19F NMR of the doublet for the C-5 fluorine of [6- H]-UMP and of the singlet for the C-5 fluorine of [6- H]-UMP (Figure 2).
1
2
and at pL 9.5-10. We have used data from NMR spectra
determined at the higher pL because of the better resolution of
peaks for the relevant protons. Table 1 reports PIEs that are the
average of values determined at two reaction times. The first
time (x min) was after ca. 95% completion of the decarboxy-
lation reaction. A second analysis at 2x min was carried out to
verify that the reaction product UMP was stable under the
reaction conditions to further OMPDC-catalyzed hydron
3
1,32
exchange,
and to provide an estimate for the uncertainty in
these product isotope effects. In every case but one, the
agreement between the PIEs determined from the products
obtained at these two reaction times is better than (5%.
Figure 2 shows a representative 19F NMR spectrum (500
MHz) at pL 7.4 of 5-FUMP obtained from the decarboxylation
of 5-FOMP (2 mM) catalyzed by R235A mutant ScOMPDC
(0.054 µM in 50/50 (v/v) HOD/DOD) at pL 7.4 and 25 °C (I )
Figure 2. 19F NMR spectrum of 5-FUMP, which is the product of
decarboxylation of 5-FOMP catalyzed by R235A ScOMPDC (0.054 µM)
at pL 7.4. The reported chemical shifts are relative to a neat trifluoracetic
0
.10, NaCl) and a reaction time of 30 min. Note that the
2
19
2
7
deuterium at C-6 of [6- H]-5-FUMP perturbs the F chemical
shift of the C-5 fluorine by 0.29 ppm. The product deuterium
isotope effect of 0.92 was determined as the ratio of the
integrated area of the doublet at δ ) 47.70 ppm (JHF ) 6 Hz)
acid external standard for which δ ) -78.5 ppm. Key: (∆) doublet at δ
1
)
47.70 ppm (JHF ) 6 Hz) due to the fluorine at [6- H]-5-FUMP; ([)
2
downfield-shifted singlet at δ ) 47.41 ppm due to the fluorine at [6- H]-
-FUMP.
5
1
due to the fluorine at [6- H]-5-FUMP and of the downfield-
DOD at pL 7.4 and 25 °C (I ) 0.10, NaCl) and a reaction time
of 30 min. The PIE of A /A
2
shifted singlet at δ ) 47.41 due to the C-5 fluorine at [6- H]-
H
D
) 1.03 ( 0.01 was calculated
5
-FUMP (Scheme 2, X ) F).
from the ratio of the integrated area of the doublet at 5.846
Table 1 reports the PIEs on decarboxylation of 5-FOMP (2
1
ppm for the C-5 hydrogen of [6- H]-UMP and of the collapsed
mM) at pL 7.4 catalyzed by wild-type ScOMPDC and by several
mutant forms of ScOMPDC. These PIEs were calculated as the
2
singlet at 5.843 ppm for the C-5 hydrogen of [6- H]-UMP. This
PIE is the average of the values determined at two different
reaction times, and the quoted uncertainty gives the range of
the two experimental values.
1
ratio A
H
/A
D
of the integrated area of C-5 fluorine of [6- H]-5-
2
FUMP and of the C-5 fluorine of [6- H]-5-FUMP determined
for spectra similar to the spectrum shown in Figure 2. The PIEs
reported in Table 2 are the average of values determined at two
reaction times. The first time (x min) was after ca. 95%
completion of the decarboxylation reaction. A second analysis
Table 1 reports product deuterium isotope effects on the
decarboxylation of OMP (2 mM) at pL 7.4 catalyzed by wild-
type OMPDC from several different organisms and by several
mutant forms of OMPDC from S. cereVisiae. These PIEs were
calculated as the ratio of the integrated area of the C-5 hydrogen
(
30) Massoud, S. S.; Sigel, H. Inorg. Chem. 1988, 27, 1447–1453.
1
2
of [6- H]-UMP and of the C-5 hydrogen of [6- H]-UMP
determined for spectra at pL 9.5-10, similar to the spectrum
shown in Figure 1B. There is good agreement ((5%) between
the PIEs calculated from spectral data at pL 7.4 (Figure 1A)
(31) Amyes, T. L.; Wood, B. M.; Chan, K.; Gerlt, J. A.; Richard, J. P.
J. Am. Chem. Soc. 2008, 130, 1574–1575.
(
32) Chan, K. K.; Wood, B. M.; Fedorov, A. A.; Fedorov, E. V.; Imker,
H. J.; Amyes, T. L.; Richard, J. P.; Almo, S. C.; Gerlt, J. A.
Biochemistry 2009, 48, 5518–5531.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 20, 2010 7021

A R T I C L E S
Toth et al.
31
Chart 1
OMP. The PIE on OMPDC-catalyzed decarboxylation is equal
to 1/ΦTS (eq 2, Scheme 4), where ΦTS is the fractionation factor
for partitioning of -H and -D between solvent LOL and the
+
ꢀ
-NL
3
group of the Lys-93 at the transition state for
36-38
decarboxylation.
The kinetic deuterium isotope effect (KIE
)
k
EH/kED, Scheme 4) on OMPDC-catalyzed decarboxylation
of OMP in 50/50 HOH/DOD is controlled by the fractionation
factor ΦEL for partitioning of -H and -D between solvent L-OL
+
and the ꢀ-NL
3
group of the Lys-93, and by ΦTS (eq 3, Scheme
4
). Values of ΦEL ≈ 1.0 have been reported for H/D fraction-
+
36,39
2 3
ation between L
equal to the KIE.
O and R-NL
,
so that the PIE of 1.0 is
(2)
PIE ) 1/ΦTS
(
kEH
kED
K
K
EH
ΦEL
KIE )
)
)
) Φ (PIE)
(3)
(
)
(
(
ED
)
EL
ΦTS
The values of PIE ) KIE ) 1.0 for decarboxylation of OMP
catalyzed by wild-type ScOMPDC, EcOMPDC, and MtOMPDC
show that there is no selectivity between the reaction of -H and
-D at the transition state for the rate-determining step. Therefore,
the zero-point energy of the bond to the transferred hydron does
not change significantly on proceeding first from the ground
+
state of 50/50 HOH/DOD to the ꢀ-NL
3
of Lys-93, and then to
at 2x min was carried out to verify that the product 5-FUMP
the transition state for the rate-determining step for the OMPDC-
catalyzed decarboxylation reaction. The absence of any signifi-
cant primary deuterium isotope effect shows that the rate-
limiting step for OMPDC catalyzed decarboxylation is strongly
uncoupled from the proton-transfer step.
was stable under the reaction conditions to further OMPDC-
3
1,32
catalyzed hydron exchange.
The agreement between the
PIEs determined for the products obtained at these two reaction
times is better than (5%.
It is interesting that essentially the same PIE is observed for
OMPDCs from S. cereVisiae, M. thermautotrophicus, and E.
coli (Table 1). These three OMPDCs show striking differences
in the size and structure of a flexible loop that interacts with
the phosphodianion of bound substrate and provides binding
Discussion
The product deuterium isotope effect on electrophilic sub-
stitution reactions of the proton in aqueous solution depends
strongly on the lifetime of the putative carbanion intermediate
4,25
3
3
energy that is utilized in catalysis of decarboxylation of OMP,
of the stepwise reaction. A PIE of 2.8 was determined for
6
-
but they otherwise exhibit a high degree of structural homology.
The results reported here show that the PIEs on these OMPDC-
catalyzed reactions are not controlled by the size of the enzyme
flexible phosphate gripper loops.
addition of the HOCH
2
CH
A). This reaction proceeds through the R-cyano-stabilized
carbanion 1, which shows a modest selectivity for protonation
2
S to acrylonitrile in water (Chart
1
8
1
3
4
by HOL compared with DOL. The sharp decease from 10 to
The PIE of 1.0 for decarboxylation of OMP is inconsistent
with a transition state for a concerted mechanism in which there
is significant electrophilic “push” by the transferred hydron to
1
.2 in the PIE on methoxide ion-catalyzed cleavage of substi-
tuted benzyltrimethylsilanes in 50/50 MeOH/MeOD (Chart 1B)
is due to the large decrease in the selectivity of the strongly
resonance-stabilized carbanion 4-NO -2 compared to the much
more unstable unsubsituted benzylic carbanion H-2. Finally,
the PIE of 1.9 determined for specific base-catalyzed retroaldol
cleavage of 1-phenylcyclopropanol reflects the stronger elec-
trophilic push of HOL compared with DOL for C-C bond
cleavage by a concerted reaction mechanism. This concerted
mechanism is enforced because the putative primary carbanion
intermediate 3 of the stepwise mechanism is far too unstable to
6
-8
displacement of CO
2
.
The observations that OMPDC cata-
2
17
lyzes the slow exchange of the C-6 hydrogen of UMP for
3
1
deuterium from solvent and of the ca. 1000-fold faster
32
exchange of the C-6 hydrogen of 5-FUMP show that OMPDC
provides strong stabilization of a bound UMP carbanion. They
provide strong support for formation of the UMP carbanion as
3
1
an intermediate of the decarboxylation reaction. The PIEs
reported in this work cannot be easily rationalized by a stepwise
reaction mechanism in which the lifetime of a UMP carbanion
reaction intermediate is sufficiently long to allow for it to
3
3,35
exist for the time of a bond vibration in water.
X-ray crystallographic analysis of the complex between
ScOMPDC and the transition-state analogue 6-hydroxyuridine
“select” between reaction with -H or -D from the enzyme. This
+
is because there is a significant discrimination between proton-
5
′-monophosphate (BMP) shows that the acidic ꢀ-NH
3
group
of the Lys-93 appears to be positioned to protonate the putative
C-6 carbanion reaction intermediate of decarboxylation of
(
(
36) Schowen, K. B.; Schowen, R. L. Methods Enzymol. 1982, 87, 551–
06.
6
37) Schowen, R. L. Prog. Phys. Org. Chem. 1972, 9, 275–332.
(
(
33) Jencks, W. P. Acc. Chem. Res. 1980, 13, 161–169.
(38) Kresge, A. J.; O’Ferrall, R. A. M.; Powell, M. F. In Isotopes in
Organic Chemistry; Buncel, E., Ed.; Elsevier: Amsterdam, 1987; pp
178-275.
34) Fishbein, J. C.; Jencks, W. P. J. Am. Chem. Soc. 1988, 110, 5075–
5
086.
(35) Thibblin, A.; Jencks, W. P. J. Am. Chem. Soc. 1979, 101, 4963–4973.
(39) Jarret, R. M.; Saunders, M. J. Am. Chem. Soc. 1986, 108, 7549–7553.
7
022 J. AM. CHEM. SOC. 9 VOL. 132, NO. 20, 2010

Product Deuterium Isotope Effects on Enzyme-Catalyzed Decarboxylation
A R T I C L E S
Scheme 4
Scheme 5
2
1
ation of carbanions by -H and -D, even when proton transfer is
strongly thermodynamically favorable.
of this activated substrate. The value of the product isotope
effect is determined by the rate constants for partitioning of the
carbanion intermediate between reaction with -H and with -D,
and it is not affected by this change in rate-determining step
for the overall decarboxylation reaction.
1
8
We propose that breakdown of OMP to form a C-6 UMP
carbanion intermediate is the rate-determining step for OMPDC-
catalyzed decarboxylation, and that hydron transfer from the
side chain of Lys-93 to this vinyl carbanion is faster than
+
The rate constant for C-NL bond rotation in water is
3
+
11 -1
movement that exchanges the positions of the N-L hydrons
similar to the value of 10
s
estimated for reorganization
and allows the carbanion to select for reaction with H or D
that exchanges the relative positions of -O and -S at a
thiobenzoate·carbocation ion pair in water. The X-ray crystal
structure of yeast OMPDC complexed with BMP shows that
the -CH -NH3 group of Lys-93 is fixed by hydrogen bonds
to the carboxylate groups of Asp-91 and Asp-96 (Scheme 5).
1
2
41
(
k
-p . k
r
, Scheme 5). The yield of [6- H]-UMP and [6- H]-
UMP products is therefore determined by the initial enrichment
+
of this acid in -H and -D (ΦEL, Scheme 4).
These results are consistent with the observed - CO
2
13
-
42
2
kinetic
isotope effect of 1.0255 on kcat/K for OMPDC-catalyzed
m
We propose that these two hydrogen bonds restrict rotation about
2
1
+
decarboxylation of OMP, which shows that C-C bond
cleavage partly limits the oVerall rate of this decarboxylation
the carbon-nitrogen bond of the CH -NL group of Lys-93,
2
3
11
-1
so that k , 10 s , and that they direct the single unliganded
hydron toward the C-6 UMP carbanion. Decarboxylation of
r
2
1,40
13
reaction.
The decrease to 1.0106 in the [ C]-KIE on kcat
/
1
3
-
K
m
for OMPDC-catalyzed decarboxylation of [6- C
2
]-5-FOMP
bound OMP (Scheme 5) then results in a deuterium enrichment
of product UMP that is essentially the same as the enrichment
is consistent with a partial change in rate-determining step, from
C-C bond cleavage to substrate binding, for decarboxylation
(
41) Richard, J. P.; Tsuji, Y. J. Am. Chem. Soc. 2000, 122, 3963–3964.
(40) Ehrlich, J. I.; Hwang, C.-C.; Cook, P. F.; Blanchard, J. S. J. Am. Chem.
(42) Miller, B. G.; Hassell, A. M.; Wolfenden, R.; Milburn, M. V.; Short,
Soc. 1999, 121, 6966–6967.
S. A Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2011–2016.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 20, 2010 7023

A R T I C L E S
Toth et al.
of CH
forward commitment for transfer of the non-hydrogen-bonded
hydron of Lys-93 to the C-6 UMP carbanion (k-p . k , Scheme
).
Effect of Changing Reactivity of Substrate and Enzyme. The
2
-NL +
3
in 50/50 HOH/DOD, because of the strong
with an increase in ΦEL from 1.0 for decarboxylation of OMP
to 1.1 for decarboxylation of 5-FOMP. This increase in ΦEL
provides evidence that the effect of the 5-fluorine at enzyme-
bound 5-FOMP is to strengthen the N-L bonds of the
r
5
+
37,38
-CH
2
-NL
3
group of Lys-93.
This may be due to an
PIE of 0.93 for OMPDC-catalyzed decarboxylation of 5-FOMP
by wild-type ScOMPDC is ca. 10% smaller than the value of
interaction between the negatiVe end of the C-F dipole and
+
the positiVely charged -CH
2
-NL
3
that stabilizes the alky-
1
.02 determined for wild-type OMPDC-catalyzed decarboxy-
lammonium ion relative to the basic amine.
lation of OMP (Table 1). Similar 5-10% differences in PIEs
for OMPDC-catalyzed decarboxylation of 5-FOMP and OMP
are also observed for the reactions catalyzed by several mutant
enzymes. These differences are reproducible and larger than the
uncertainty in the individual PIEs.
The PIEs on decarboxylation of OMP and 5-FOMP catalyzed
by OMPDC show a remarkable insensitivity to mutation of
amino acid residues that are essential for the observation of
robust enzymatic activity (Table 1). In other words, these
mutations affect the stability of the rate-determining transition
state for decarboxylation but do not affect the relative yield of
H- and D-labeled reaction products. The results are consistent
The addition of the 5-F to OMP causes only small changes
7
7
-1 -1
in kcat/K
(
m
(from 1.3 × 10 to 2.6 × 10 M s ) and in kcat
-1
23
from 20 to 130 s ) for ScOMPDC-catalyzed decarboxylation.
with the mechanism shown in Scheme 5, where k-p . k for
r
However, the full effect of the 5-fluorine substituent on the
microscopic rate constant for decarboxylation of bound substrate
is not observed, because binding of 5-FOMP to OMPDC is
all mutant enzyme-catalyzed reactions. This is simply rational-
ized. The active-site residues examined all lie distant from Lys-
93, so their mutation is not expected to affect the barrier to kr
+
strongly rate-determining for kcat/K
-FUMP is strongly rate-determining for kcat
addition of the 5-fluorine to OMP causes a much larger, 200-
m
, and release of product
for rotation about the carbon-nitrogen bond of the -CH -NL
2
3
23
5
.
By comparison,
group of Lys-93. The mutations all cause an increase in the
barrier for decarboxylation of OMP to form the C-6 UMP
carbanion. This presumably reflects an increase in the barrier
to formation of the carbanion intermediate, which should be
accompanied by a decrease in the activation barrier to carbanion
5
-1 -1
fold increase in kcat/K
1
m
(from 610 to 1.2 × 10 M s , Table
-1
43
) and 270-fold increase in kcat (from 1.2 to 320 s ) for
decarboxylation of OMP catalyzed by the R235A mutant of
ScOMPDC. This shows that the 5-fluorine provides ca. 3 kcal/
mol stabilization of the transition state for OMPDC-catalyzed
decarboxylation when the breakdown of enzyme-bound 5-FOMP
is the rate-determining step. This substituent provides a similar
stabilization of the UMP carbanion intermediate of the OMPDC-
4
4
protonation (increase in k , Scheme 5) that will favor
-
p
observation of a unitary PIE.
There is a striking contrast between the large conformational
flexibility of OMPDC that is suggested by the differences in
4
2
the structures for the free and liganded enzyme and the
3
2
+
catalyzed deuterium exchange reaction of UMP.
rotational rigidity of the -CH -NL3 group of Lys-93 that is
2
Stabilization of the OMPDC-bound UMP carbanion by the
hydrogen-bonded to carboxylate groups of Asp-91 and Asp-96
4
2
5
5
-fluorine substituent should lead to a decrease in k-p (Scheme
) for protonation of this intermediate. The PIE of 0.93 for
(Scheme 5). We note that the most efficient catalysis of
decarboxylation will be observed when there is the maximum
increase in stabilizing electrostatic interactions between the
4
4
decarboxylation of 5-FOMP catalyzed by wild-type ScOMPDC
Table 1) shows not only that this decrease in k-p is not
sufficiently large to affect the inequality k-p . k (Scheme 5)
+
(
-CH -NL3 group of OMPDC and the bound substrate/
2
r
intermediate on proceeding from the Michaelis complex to the
C-6 UMP carbanion reaction intermediate. Therefore, one role
that was proposed for partitioning of the UMP vinyl carbanion,
but also that the PIE has become smaller than the limiting KIE
of 1.0 for a reaction where there the is no movement of hydrogen
on moving from reactant to transition state. We propose that
the PIE of 0.93 is observed because the deuterium enrichment
+
for “clamping” the -CH -NL3 side chain of Lys-93 may be
2
to position this amino acid side chain to minimize its electrostatic
-
interactions with the -CO2 of OMP in the Michaelis complex
and to maximize the interactions with the developing C-6 UMP
4
5,46
of the product 5-FUMP is the same as the enrichment of
carbanion at the transition state for decarboxylation.
This
+
CH
2
-NL
3
at the OMPDC ·5-FOMP Michaelis complex. This
may involve a shift in the position of the side chain that is
enrichment is measured by the fractionation factor ΦEL (PIE )
KIE/ΦEL, Scheme 4). The smaller PIE on wild-type ScOMPDC-
catalyzed decarboxylation of 5-FOMP (0.93 ( 0.02) compared
to decarboxylation of OMP (1.02 ( 0.02, Table 1) is consistent
coupled in some way to decarboxylation.
Acknowledgment. We acknowledge the National Institutes of
Health (Grant GM39754 to J.P.R. and Grant GM65155 to J.A.G.)
for generous support of this work.
(
(
(
43) Barnett, S. A., University at Buffalo, 2009.
Supporting Information Available: Protocol for preparation
of Y217A mutant OMPDC. This information is available free
of charge via the Internet at http://pubs.acs.org.
44) Hammond, G. S. J. Am. Chem. Soc. 1955, 77, 334–338.
45) Hur, S.; Bruice, T. C Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9668–
9
673.
46) Warshel, A.; Strajbl, M.; Villa, J.; Florian, J. Biochemistry 2000, 39,
4728–14738.
(
1
JA102408K
7
024 J. AM. CHEM. SOC. 9 VOL. 132, NO. 20, 2010