Product deuterium isotope effect for orotidine 5′-monophosphate decarboxylase: Evidence for the existence of a short-lived carbanion intermediate

Article abstract of DOI:10.1021/ja076222f

A product isotope effect (PIE) of 1.0 was obtained from 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) H₂O/D₂O catalyzed by orotidine 5′-monophosphate decarboxylase. This unitary product isotope effect 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. The complete lack of selectivity for the reaction of solvent H and D that is implied by the value of PIE = 1.0 may be enforced by restricted motion of the NL₃⁺ group of the side-chain of Lys-93 that has been proposed to protonate a vinyl carbanion intermediate. Copyright

Full text of DOI:10.1021/ja076222f

Published on Web 10/05/2007
Product Deuterium Isotope Effect for Orotidine 5′-Monophosphate
Decarboxylase: Evidence for the Existence of a Short-Lived Carbanion
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 August 17, 2007; E-mail: jrichard@chem.buffalo.edu; j-gerlt@uiuc.edu
We report that equal yields of [6-1H]-uridine 5′-monophosphate
Scheme 2
(50%) and [6-2H]-uridine 5′-monophosphate (50%) are obtained
from the decarboxylation of orotidine 5′-monophosphate (OMP)
catalyzed by orotidine 5′-monophosphate decarboxylase in a solvent
of 50/50 (v/v) H2O/D2O. This observation of an unusually small
product isotope effect of unity eliminates a proposed mechanism
in which proton transfer from Lys-931 to C-6 provides electrophilic
push to the loss of CO2 from OMP in a concerted reaction.2,3 It
provides evidence that proton transfer from the ammonium cation
side chain of Lys-93 to a vinyl carbanion intermediate is faster
than the bond rotation that exchanges the positions of the acidic
N-L+ hydrons of this side chain.
Orotidine 5′-monophosphate decarboxylase (OMPDC) is a
represent a secondary solvent kinetic isotope effect (SKIE). By
contrast, a product isotope effect (PIE) determined in experiments
in which H and D in a mixed solvent of H2O/D2O compete for
reaction with enzyme-bound OMP to form UMP labeled at C-6
(Scheme 2) would provide insight into the changes in bonding at
the transferred hydron that occur on proceeding to the transition
remarkable enzyme because it employs no metal ions or other
cofactors but yet effects an enormous 1017-fold acceleration of the
chemically very difficult decarboxylation of OMP to give uridine
5′-monophosphate (UMP).4,5 It has been shown that a large fraction
of the enzymatic rate acceleration results directly from utilization
of the intrinsic binding energy of the remote nonreacting 5′-
phosphodianion group of OMP in transition state stabilization.6 The
decarboxylation reaction is often proposed to proceed in two steps
through a vinyl carbanion intermediate (Scheme 1). However, it
has also been suggested that this unstable intermediate might be
avoided in a concerted reaction in which decarboxylation and proton
transfer to C-6 occur in a single step.2,3
state for the product-determining step.10 PIEs are more precise and
easier to interpret than SKIEs determined as the ratio of rate
constants for reactions in H2O and D2O because (1) there are no
complications from any secondary SKIE when the H- and D-labeled
products are formed in the same mixed H2O/D2O solvent and (2)
there are no errors due to differences in the conditions for separate
reactions in H2O and D2O, such as enzyme concentration, temper-
ature, and pL.
The product distribution for the decarboxylation of OMP
catalyzed by OMPDC in 50/50 (v/v) H2O/D2O was determined by
1H NMR spectroscopy at 500 MHz. Figure 1 shows the partial 1H
NMR spectrum of UMP obtained from the decarboxylation of
OMP (2 mM) catalyzed by OMPDC from S. cereVisiae (C155S
mutant, 24 nM, 1 h, >90% reaction) in 50/50 (v/v) H2O/D2O at
pL 7.3 and 25 °C (I ) 0.10, NaCl).11,12 The value of PIE ) 1.0
was calculated using eq 1, where AH is the integrated area of the
doublet due to the C-6 proton of [6-1H]-UMP (7.990 ppm), and
AD is the integrated area of the singlet due to the C-5 proton of
[6-2H]-UMP (5.865 ppm).13 By comparison, PIEs of 7.3-8.1 for
proton transfer to ring-substituted aryl vinyl ethers from lyonium
ion in 50/50 (v/v) H2O/D2O have been reported recently.10
Scheme 1
Experimental and computational studies on OMPDC have
focused largely on the partly rate-determining and highly unfavor-
able loss of CO2 from OMP.7-9 There are few data pertaining to
the proton transfer to C-6 of the pyrimidine ring. Experimental
characterization of this proton-transfer step is essential for insight
into the existence and lifetime of the putative enzyme-bound vinyl
carbanion intermediate.
OMPDC catalyzes incorporation of a hydron from solvent into
the UMP product and it has been reported that the decarboxylation
of saturating OMP is 30% faster in H2O than in D2O.7 While the
origin of this solvent isotope effect on kcat is unclear, it may
PIE ) AH/AD
(1)
We used similar procedures to determine values of PIE ) 1.0 for
decarboxylation of OMP (2 mM) catalyzed by OMPDC from both
E. coli (40 nM) and M. thermoautotrophicum (40 nM) in 50/50
(v/v) H2O/D2O at pL 7.3 and 25 °C (I ) 0.10, NaCl). The essentially
identical PIEs determined for OMPDC from different sources is
significant, because these enzymes exhibit somewhat different
architectures at their active sites.2,9a,14,15
The value of PIE ) 1.0 for the OMPDC-catalyzed decarboxy-
lation of OMP in 50/50 (v/v) H2O/D2O shows that the deuterium
enrichment of the hydron used to protonate OMP or an intermediate
carbanion at the reaction transition state (50%) is the same as that
† University at Buffalo.
# University of Illinois.
9
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J. AM. CHEM. SOC. 2007, 129, 12946-12947
10.1021/ja076222f CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
it may be a step that orients an N-L+ bond of Lys-93 into a “reactive
position” where hydron transfer to a vinyl carbanion intermediate
can occur. In both cases the PIE of 1.0 requires that the chemical
step of hydron transfer to the carbanion be faster than any molecular
motion that allows its discrimination between reaction with H and
D at the NL3+ group of Lys-93.17 We therefore propose that hydron
transfer from the side chain of Lys-93 to a vinyl carbanion
intermediate (kp) is faster than any movement that exchanges the
positions of the N-L+ hydrons and which would allow the
carbanion to select for reaction with H or D.17 In water, the rate
constant for such a step is ca. 1011 s-1 20
.
The X-ray crystal structure of yeast OMPDC complexed with
6-hydroxyuridine 5′-monophosphate shows that the CH2-NH3
Figure 1. Partial 1H NMR spectrum (500 MHz) of UMP from decarboxy-
lation of OMP (2 mM) catalyzed by OMPDC from S. cereVisiae (24 nM)
in 50/50 (v/v) H2O/D2O at pL 7.3 and 25 °C: (1) doublet due to the C-6
proton of [6-1H]-UMP; (b) doublets (not resolved) due to the anomeric
protons of [6-1H]-UMP and [6-2H]-UMP; (×) doublet due to the C-5 proton
of [6-1H]-UMP; ([) singlet due to the C-5 proton of [6-2H]-UMP.
+
group of Lys-93 is anchored by two hydrogen bonds to the
carboxylate groups of Asp-91 and Asp-96 that are proposed to direct
the third ammonium hydron of Lys-93 toward the putative vinyl
carbanion intermediate.15 These hydrogen bonds should also restrict
+
rotation about the carbon-nitrogen bond of the terminal CH2-NL3
of the 50/50 (v/v) H2O/D2O solvent. The product-determining step
group of Lys-93 (krot , 1011 s-1). This would favor the observed
unselective proton transfer from the remaining free (non-hydrogen-
bonded) hydron to a vinyl carbanion intermediate.
+
is thought to be proton transfer from the NL3 group of the side
chain of Lys-93 to OMP or to a reaction intermediate (Scheme
3).15 Values of φNL3+ ≈ 1.0 have been reported for the H/D
fractionation between L2O and R-NL3+, so that the deuterium
enrichment of the NL3+ group of Lys-93 should be similar to that
of the solvent L2O.16 Therefore the PIE of 1.0 is essentially equal
to the primary kinetic isotope effect for reaction of the H- and
D-labeled NL3+ group of Lys-93 to form [6-1H]-UMP and [6-2H]-
UMP.
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.
References
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Scheme 3
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state for the product-determining step,17a and there is no precedent
for PIEs as small as 1.0 when carbanion protonation is the product-
determining step.17a,18 The observed PIE of 1.0 requires that all of
the zero-point energy present in the N-L+ bonds of Lys-93 be
maintained at the transition state for the step that determines whether
the UMP product is labeled at C-6 with H or D. This PIE is not
consistent with a mechanism in which proton transfer from Lys-
93 to C-6 of OMP provides electrophilic push to the loss of CO2
in a concerted reaction that avoids formation of an unstable vinyl
carbanion intermediate (bottom pathway, Scheme 3).2,3,19
We suggest that the essentially statistical yields of [6-1H]-UMP
and [6-2H]-UMP from the OMPDC-catalyzed decarboxylation of
OMP are established at a step that occurs prior to hydron transfer
to a vinyl carbanion intermediate. This could be the decarboxylation
step, if an N-L+ bond of Lys-93 is already correctly positioned to
deliver a hydron to a vinyl carbanion (kdc, Scheme 3). Alternatively
(13) Control experiments showed that AH/AD (eq 1) remains constant over a
period of ca. 20 hours. The variation in this ratio determined from
integration of different NMR spectra is less than 3%.
(14) Harris, P.; Poulsen, J.-C. N.; Jensen, K. F.; Larsen, S. J. Mol. Biol. 2002,
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(19) The primary 13C KIE on OMPDC-catalyzed decarboxylation of OMP
labeled at the carboxylate carbon decreases from 1.043 for reaction in
H2O to 1.034 for the 25% slower reaction in D2O (ref 7), which shows
that decarboxylation is less rate-determining in D2O than in H2O. This
result is not easily rationalized by a mechanism in which proton transfer
from Lys-93 to C-6 of OMP is concerted with the loss of CO2 because
the change from H2O to D2O should raise the barrier to a reaction in
which proton transfer is concerted with loss of CO2, as a result of a normal
primary KIE. This would cause the loss of CO2 to become more rate-
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