Evidence for a stepwise mechanism of OMP decarboxylase [16]

Full text of DOI:10.1021/ja990737s

6966
J. Am. Chem. Soc. 1999, 121, 6966-6967
Evidence for a Stepwise Mechanism of OMP
Decarboxylase
Joel I. Ehrlich,† Chi-Ching Hwang,‡ Paul F. Cook,‡ and
John S. Blanchard*,†
Department of Biochemistry
Albert Einstein College of Medicine
Bronx, New York 10461
Figure 1. Reaction catalyzed by ODCase, and the putative ylide
intermediate.
Department of Chemistry and Biochemistry
UniVersity of Oklahoma
Table 1. Kinetic Isotope Effects for E. coli ODCase
Norman, Oklahoma 73019
isotope effect
value
13(V/K)H O
13C KIE in H2O
1.043 ( 0.003
1.034 ( 0.002
1.3 ( 0.2
ReceiVed March 8, 1999
2
13(V/K)D O
13C KIE in D2O
D2O solvent KIE
2
D O(V/K)
2
Orotidine 5′-monophosphate decarboxylase (ODCase, EC
4.1.1.23) catalyzes the conversion of orotidine 5′-monophosphate
(OMP) to uridine 5′-monophosphate (UMP) in the final step of
de novo pyrimidine biosynthesis (Figure 1). The enzyme catalyzes
the most “proficient” enzymatic reaction described to date,1 with
the enzyme-catalyzed V/K value exceeding the spontaneous
reaction by a factor of 1023. The chemical mechanism of the
enzyme-catalyzed reaction, however, has remained enigmatic, and
a number of possible mechanisms have been proposed over the
years. One mechanistic model for ODCase action invokes
protonation of the C2 carbonyl to generate a zwitterionic nitrogen
ylide intermediate (Figure 1) upon decarboxylation in a subsequent
step.2 This mechanism is consistent with the very strong inhibition
(Ki ≈ 10-11 M) of ODCase by 1-â-D-ribofuranosylbarbituric acid
5′-phosphate.3 The magnitude of the primary 13C kinetic isotope
effect (KIE) (1.0247 ( 0.0008 at pH 6.8, 25 °C)4 of the yeast
enzyme suggests that decarboxylation is the rate-limiting step.
Recently, Lee and Houk5 have proposed a novel mechanism
for ODCase on the basis of quantum mechanical calculations.
They noted that the decarboxylation of C4-keto protonated OMP
yields a carbene product that is more stable than the ylide which
results from C2-keto protonated OMP. Analysis of gas-phase ∆Hq
and proton affinities supported the suggestion that protonation at
the C4-keto oxygen of OMP was more favorable. They further
argued that protonation should be concerted with decarboxylation,
since the departure of CO2 would raise the effective pKa of the
C4 ketone such that it could accept a proton from a putative
active-site lysine residue6 with apparent pKa ≈ 7.4
Figure 2. Contour plot representing the set of solutions for eqs 1-3
that satisfy the measured KIEs for ODCase. The axis ranges are
abbreviated such that only the chemically reasonable solutions are
displayed, as described in the text.
The measurement of multiple KIEs can be used to distinguish
whether an enzymatic reaction occurs via a stepwise or a concerted
mechanism.7 To test the proposal that the decarboxylation of OMP
is concerted with protonation of the substrate, the D2O solvent
KIE and 13C primary KIEs in H2O and D2O were measured for
Escherichia coli ODCase8,9 (Table 1). Solvent KIEs were
ation and proton transfer12 occur in separate steps. Proton transfer
must occur prior to decarboxylation, since decarboxylation is
measured in a standard spectrophotometric assay,10 and yielded
(8) The DNA sequence encoding E. coli ODCase (pyrF gene) was amplified
from genomic DNA by PCR (Perkin-Elmer), digested with NdeI and BamH1
(New England BioLabs), and ligated into the expression vector pET23a
(Novagen). E. coli BL21(DE3) cells containing the resulting pET:pyrF plasmid
D O(V/K) of 1.3 ( 0.2. Carbon isotope effects on V/K were
2
a
determined by measuring the ratio of 13C/12C in product CO2 by
isotope ratio mass spectrometry.11 The 13C kinetic isotope effect
is 1.043 ( 0.003 in H2O, and decreases to 1.034 ( 0.002 in D2O.
This outcome is diagnostic of a mechanism in which decarboxyl-
were cultured in Luria Broth + 50 mg/L carbenicillin at 37 °C until A600
)
0.6-0.8. Isopropyl-â-D-thiogalactoside was added to a final concentration of
100 µM, and cells were harvested after an additional 3 to 4 h. The cell pellet
was resuspended (1:1 weight:volume) in 20 mM TEA pH 7.8, 1 mM DTT,
Complete protease inhibitors (Boehringer-Mannheim), 1 mg/mL lysozyme,
frozen at -20 °C overnight and thawed at room temperature. The sample
was disrupted by sonication and centrifuged. Streptomycin sulfate (pH 8) was
added to the supernatant to a final concentration of 1% to precipitate nucleic
acids. Following centrifugation, the supernatant (approximately 100 mL) was
dialyzed 3-4 h against 12 L 20 mM TEA pH 7.8, 1 mM DTT, 50 mM NaCl,
and ultracentrifuged (30K rpm). ODCase in the resulting supernatant was then
purified to homogeneity by fast protein liquid chromatography (FPLC) on
FastQ anion-exchange and phenyl-sepharose hydrophobic interaction columns
(Pharmacia).
* To whom correspondence may be addressed. Telephone: (718) 430-
3096. Fax: (718) 430-8565. E-mail: blanchar@aecom.yu.edu.
† Albert Einstein College of Medicine.
‡ University of Oklahoma.
(1) Radzicka, A.; Wolfenden, R. Science 1995, 267, 90-93.
(2) Beak, P.; Siegel, B. J. Am. Chem. Soc. 1976, 98, 3601-3606.
(3) Levine, H. L.; Brody, R. S.; Westheimer, F. H. Biochemistry 1980, 19,
4994-4999.
(4) Smiley, J. A.; Paneth, P.; O’Leary, M. H.; Bell, J. B.; Jones, M. E.
Biochemistry 1991, 30, 6216-6223.
(5) Lee, J. K.; Houk, K. N. Science 1997, 276, 942-945.
(6) Smiley, J. A.; Jones, M. E. Biochemistry 1992, 31, 12162-12168.
(7) Hermes, J. D.; Roeske, C. A.; O’Leary, M. M.; Cleland, W. W.
Biochemistry 1982, 21, 5106-5114.
(9) E. coli ODCase was inactivated (90%) by overnight dialysis against
0.5 mM EDTA. This is consistent with the recent suggestion (Miller, B. G.;
Traut, T. W.; Wolfenden, R. J. Am. Chem. Soc. 1998, 120, 2666-2667) that
the enzyme requires a metal cofactor.
10.1021/ja990737s CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/14/1999

Communications to the Editor
J. Am. Chem. Soc., Vol. 121, No. 29, 1999 6967
Table 2. Mechanistic Parameters for E. coli ODCase
irreversible, and subsequent steps are not included in the
expression for V/K. A minimal mechanism is thus
parameter
lower limit
upper limit
a
c
k3/k2
k5/k4
0m
4.3m
0.1m
1.8m
1.047m
0.63m
5.0a
D Ok3
intrinsic D2O KIE
2
13k5
intrinsic 13C KIE
1.07a
a Assumed constraint based upon chemical considerations. m Math-
ematical limit within the experimental results and chemical constraints.
where substrate binding (k1, k2) is followed first by reversible
substrate protonation (k3, k4),13 and finally by irreversible decar-
boxylation (k5). The isotope effects for this mechanism are
described by the following equations:
D Ok3 + a + D OKeq/c
1 + a + 1/c
2
2
D O(V/K) )
(1)
(2)
(3)
2
13k5 + c(1 + a)
13(V/K)H O
)
)
2
1 + c(1 + a)
Figure 3. Proposed stepwise mechanism of ODCase, with protonation
at O4 followed by decarboxylation to the carbene.
13k + c D Ok4 + a/D OKeq
2
2
(
)
5
13(V/K)D O
1 + c D Ok4 + a/D OKeq
2
2
2
rate-limiting, such that the protonated intermediate preceding
decarboxylation tends to deprotonate between 62 and 91% (1.6
e 1/c e 10) of the time, are possible. Either a partially or barely
rate-limiting protonation step is compatible with these results.
These data suggest a modification of the Houk mechanism5
for ODCase as shown in Figure 3.16 In this mechanism, initial
C4 keto protonation by an essential enzymatic lysine residue
generates the C6 cation. Since protonation concomitant with
decarboxylation is excluded experimentally, we suggest that this
thermodynamically unfavorable step may instead be promoted
by a hydrogen bonding network to the C4 keto oxygen of the
orotate moiety. For this substrate, such a network has been
observed and implicated in the mechanism of the enzyme which
immediately precedes ODCase in the pyrimidine biosynthetic
pathway, orotate phosphoribosyl transferase.17 Decarboxylation
of the C6 cation then generates the carbene proposed by Lee and
Houk in the rate-limiting step.
(
)
13(V/K)H O - 1
2
D O(V/K)
2
)
(4)
13(V/K)D O - 1
K
D2O
eq
2
where D Ok3 and D Ok4 are the intrinsic D2O solvent KIEs in the
2
2
forward and reverse directions, 13k5 is the intrinsic 13C KIE, D OKeq
2
2
2
)
D Ok3/D Ok4 is the D2O solvent equilibrium isotope effect, a )
k3/k2, and c ) k5/k4.14 These equations are not independent, and
thus a unique analytical solution does not exist. However, the set
of solutions can be computed15 and represented as three-
dimensional surfaces.
Inspection of contour plots of c and D Ok3 over chemically
2
reasonable ranges for 13k5 and a (Figure 2) identified an upper
limit of 0.63 for c and a lower limit of 1.8 for D Ok3. When the
2
intrinsic solvent KIE D Ok3 is restricted to values e5.0, all solutions
2
with a g 4.3 and c e 0.10 are excluded, and 13k5 must be at least
1.047. This value compares favorably with the 13C KIE of 1.0494
( 0.0006 exhibited by the yeast enzyme at pH 4.0.4 The limits
on these mechanistic parameters are summarized in Table 2.
The multiple isotope effects measured for E. coli ODCase are
thus consistent exclusively with a stepwise mechanism. As Figure
2 illustrates, only mechanisms in which decarboxylation is highly
Acknowledgment. This work was supported by the National Institutes
of Health Grants GM33449 (J.S.B.) and GM50872 (P.F.C.)
Supporting Information Available: Experimental data for the
determination of D O(V/K) and 13(V/K)L O; 3-Dimensional and contour plots
2
2
of c and D Ok3 vs {13k5, a}; a plot of decarboxylation commitment factors
2
vs 13k5 (PDF). This material is available free of charge via the Internet at
http://pubs.acs.org.
(10) (a) ODCase was assayed by monitoring the decrease in A280 (ꢀ ) 1350
M-1 cm-1) as OMP is converted to UMP.10b Reactions contained OMP,
enzyme, 20 mM Hepes pL 7.5, and 0.5 mM DTT at room temperature. (b)
Brody, R. S.; Westheimer, F. H. J. Biol Chem. 1979, 254, 4238-4244.
(11) (a) The method for measuring 13(V/K) by collection and isotope ratio
analysis of product CO2 is described by O’Leary.11b All reactions contained
2 mM OMP, 20 mM Hepes pL 7.5, and 0.5 mM DTT at room temperature in
sealed vacuum flasks, and were sparged overnight with N2 to remove all
contaminating CO2 prior to addition of enzyme. D2O was previously sparged
with CO2 in the presence of carbonic anhydrase to exchange excess 18O. Two
100% conversion (20 mL) and three partial conversion (60 mL) reactions
were performed in H2O and D2O. Fractional conversion of OMP to UMP
was monitored in aliquots spectrophotometrically,10 and reactions were
quenched by the addition of concentrated sulfuric acid to pH < 1. Product
CO2 was purified by three cycles of high-vacuum distillation though dry ice-
ethanol and liquid N2 traps prior to analysis by isotope ratio mass spectrometry.
(b) O’Leary, M. H. Methods Enzymol. 1980, 64, 83-104.
(12) While other schemes (e.g., solvent-sensitive conformational change
preparatory to decarboxylation) are also consistent with these data, we retain
the pre-protonation of substrate model suggested by previous studies.2-7
(13) In this derivation, the terms k3 and k4 represent “net” rate constants
that describe the entire solvent-sensitive portion of the reaction coordinate,
which may formally consist of a single or multiple microscopic steps.
(14) The forward commitment factor (partition ratio for the enzyme form
competent to undergo decarboxylation), (k5/k4)(1 + k3/k2), is divided into an
internal commitment factor (k5/k4) and an external commitment factor (k5k3/
k3k2).
JA990737S
D2O
(15) (a) According to eq 4 and the experimental results,
K ) 1.03 (
eq
0.16. This suggests that the solvent equilibrium isotope effect is, in fact, unity.
This value is not unexpected, since the fractionation factors for N-L and
O-L are unity;15b therefore, D OKeq ) 1 was used in the calculations. An array
2
of values for 13k5 and a, representing the complete range of chemically
reasonable solutions, was generated. A minimum value of 1.043 was selected
for 13k5, corresponding to the observed KIE (the observation of partially rate-
limiting D2O effects requires that 13k5 be higher than this). The upper limit
given for 13k5 was 1.07, approximating the largest known values for enzymatic
decarboxylations.15c,d The forward commitment factor a was given an initial
range of 0 to 5. Equation 2 was used to calculate c for each point in the array,
and D Ok3 was calculated with eq 1 from the resulting {13k5, a, c} array. The
2
final {13k5, a, c, D Ok3} array satisfied eqs 1, 2, and 3, and yielded values for
2
the forward commitments for decarboxylation smaller than unity, as expected.
(b) Quinn, D. M.; Sutton, L. D. In Enzyme Mechanism from Isotope Effects;
Cook, P. F., Ed.; CRC Press: Boca Raton, Florida, 1991; 73-126. (c) Karsten,
W. E.; Gavva, S. R.; Park S. H.; Cook P. F. Biochemistry 1995, 34, 3253-
60. (d) Grissom, C. B.; Cleland, W. W. Biochemistry 1988, 27, 2927-34.
(16) The Beak and Siegel mechanism2 with protonation occurring at the
C2 carbonyl is also consistent with these results. We elect to interpret the
data in the context of the Houk mechanism on the basis of its more favorable
predicted thermodynamics.
(17) Tao, W.; Grubmeyer, C.; Blanchard, J. S. Biochemistry 1996, 35, 14-
21.