The effective molarity of the substrate phosphoryl group in the transition state for yeast OMP decarboxylase

Article abstract of DOI:10.1016/j.bioorg.2004.08.005

The second order rate constant (k_cat/K_m) for decarboxylation of orotidine by yeast OMP decarboxylase (ODCase), measured by trapping ¹⁴CO₂ released during the reaction, is 2 × 10^-4 M^-1 s^-1. This very low activity may be compared with a value of 3 × 10⁷ M^-1 s^-1 for the action of yeast OMP decarboxylase on the normal substrate OMP. Both activities are strongly inhibited by 6-hydroxy UMP (BMP), and abrogated by mutation of Asp-96 to alanine. These results, in conjunction with the binding affinity of inorganic phosphate as a competitive inhibitor (K_i = 7 × 10^-4 M), imply an effective concentration of 1.1 × 10⁹ M for the substrate phosphoryl group in stabilizing the transition state for enzymatic decarboxylation of OMP. The observed difference in rate (1.5 × 10¹¹-fold) is the largest effect of a simple substituent that appears to have been reported for an enzyme reaction.

Full text of DOI:10.1016/j.bioorg.2004.08.005

Bioorganic Chemistry 33 (2005) 45–52
www.elsevier.com/locate/bioorg
The effective molarity of the substrate
phosphoryl group in the transition state
for yeast OMP decarboxylase
Annette Sievers, Richard Wolfenden*
Department of Biochemistry and Biophysics, University of North Carolina,
Chapel Hill, NC 27599-7260, USA
Received 12 July 2004
Available online 12 October 2004
Abstract
The second order rate constant (kcat/K
m
) for decarboxylation of orotidine by yeast OMP
1
4
decarboxylase (ODCase), measured by trapping CO2 released during the reaction, is
ꢀ4
ꢀ1 ꢀ1
7
ꢀ1 ꢀ1
s
2
· 10
M
s
. This very low activity may be compared with a value of 3 · 10 M
for the action of yeast OMP decarboxylase on the normal substrate OMP. Both activities
are strongly inhibited by 6-hydroxy UMP (BMP), and abrogated by mutation of Asp-96 to
alanine. These results, in conjunction with the binding affinity of inorganic phosphate as a
ꢀ
4
9
competitive inhibitor (K
i
= 7 · 10 M), imply an effective concentration of 1.1 · 10 M for
the substrate phosphoryl group in stabilizing the transition state for enzymatic decarboxyl-
ation of OMP. The observed difference in rate (1.5 · 10 -fold) is the largest effect of a simple
substituent that appears to have been reported for an enzyme reaction.
1
1
ꢀ
2004 Elsevier Inc. All rights reserved.
Keywords: OMP; OMP decarboxylase; ODCase; Orotidine; Connectivity effect; Effective molarity;
Transition state; Binding discrimination
*
Corresponding author. Fax: +1 919 966 2852.
E-mail address: water@med.unc.edu (R. Wolfenden).
0
045-2068/$ - see front matter ꢀ 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2004.08.005

46
A. Sievers, R. Wolfenden / Bioorganic Chemistry 33 (2005) 45–52
1
. Introduction
Comparison of an enzymeÕs activity on its usual substrate, with its activity on a
truncated version of the substrate that is presumably capable of entering the active
site without steric hindrance, has shown that the binding of groups remote from the
site of substrate transformation sometimes plays a prominent role in enzyme action
[
1]. Hexokinase, for example, is virtually inactive in catalyzing the hydrolysis of
ATP, despite the ability of water to enter any cavity that can accommodate glucose,
the normal phosphoryl acceptor [2]. A rate loss upon truncation of the substrate can
be explained by utilization of binding forces to overcome the loss of translational,
rotational, and internal entropy [3]. Thus the free energy of binding of a substrate
A–B by an enzyme, compared with the sum of free energies of binding of its compo-
nent parts A and B, would be expected differ by the rotational, translational, and
internal entropies of the molecules. Comparisons of this kind are of theoretical
and practical interest, in view of the potential magnitude of connectivity effects in
determining the binding affinities of substrates in the ground state and transition
state, and of protein–ligand interactions in general.
With a half-time of 78 million years in neutral solution at 25 ꢁC, decarboxylation
of OMP (Fig. 1) is one of the more difficult reactions in biology. Yeast OMP decar-
1
7
boxylase (ODCase) enhances the rate of this reaction by a factor of 10 [4]. The
crystal structures of OMP decarboxylase from yeast and several species of bacteria
reveal numerous interactions between the active site and the ribose and phosphoryl
groups of nucleotide inhibitors, but few enzyme interactions with the pyrimidine ring
where decarboxylation occurs [5–8]. The complex network of interactions between
the active site and the phosphoryl group accords with the finding that the binding
affinities of inhibitory nucleosides fall 3–6 orders of magnitude short of the binding
affinities of the corresponding nucleotides [9].
In earlier work on yeast ODCase no decarboxylation of orotidine or orotic acid
could be detected using a UV assay [9]. In the present work, we used a more sensitive
0
radioassay to assess the contribution that the substrateÕs 5 -phosphoryl group makes
to kcat/K for yeast ODCase, and to this enzymeÕs corresponding affinity for the al-
m
tered substrate in the transition state. Here we report the reduction in rate that results
0
from removal of the 5 -phosphoryl group of OMP, and the effective molarity of this
group at the active site in the transition state for decarboxylation (see Table 1).
Fig. 1. OMP decarboxylase catalyzes the removal of the 6-carboxylate group from the pyrimidine ring of
0
orotidine 5 -monophosphate. The effect of the phosphoryl group (red) was studied in this work.

A. Sievers, R. Wolfenden / Bioorganic Chemistry 33 (2005) 45–52
47
Table 1
Comparison of orotidine and OMP decarboxylation by yeast OMP decarboxylase
ꢀ1
ꢀ1
ꢀ1 ꢀ1
(M s )
k
cat (s
)
K
m
(M
)
kcat/K
m
ꢀ
7
7
OMP
Orotidine
22
P7 · 10
7 · 10
3 · 10
2 · 10
ꢀ6
ꢀ2
ꢀ4
P3 · 10
2
. Materials and methods
1
.1. Synthesis of [ C]orotidine from 5-Br uridine
4
2
Adopting a procedure similar to that used earlier for the synthesis of OMP [10], 5-
1
4
bromouridine (20 mg) and Na CN (0.9 mg) were dissolved in DMSO (3 mL) and
allowed to react at room temperature for 48 h. After removal of DMSO by rotary
evaporation, KOH (1 mL, 3.4 M) was added to the crude 6-CN uridine and the mix-
ture was heated to 60 ꢁC for 4 h. After neutralization, the sample was purified over a
ꢀ
Dowex 2-Cl anion exchange column and eluted with HCl (0.1 M). The product ob-
tained in an overall yield of 56% was identical with authentic orotidine in its UV
0
absorption (kmax = 267) [11] and H NMR spectrum (d 5.55 ppm (1 H), d
0
5
.75 ppm (5 H), and d 4.32 (3 H)) [12].
2
.2. Source of enzyme
All experiments were performed with the C155S mutant of OMP decarboxylase.
Mutation of residue Cys-155 to serine results in a more stable protein than WT OD-
Case, but does not affect ligand binding, substrate turnover, or the overall structure
of the enzyme (unpublished data). Both C155S and D96A/C155S yeast OMP decar-
boxylase were expressed in Escherichia coli and purified by anion exchange chroma-
tography over a Poros IIQ (Perspective Biosystems) and HR 10/10 MonoQ
(
Amersham Pharmacia Biotech) column as previously described [13].
.3. Assay of orotidine decarboxylation
The decarboxylation of orotidine was measured by trapping the product CO on
2
1
4
2
KOH-soaked filter paper in a scintillation vial sealed by a rubber septum [14]. A typ-
ical reaction mixture (0.5 mL), containing OMP decarboxylase (1–5 mg/mL),
1
4
[
C]orotidine (1–9 mM), and Mops (100 mM, pH 7.2), was incubated at 23 C for
–48 h. 10% Glycerol was added to the reaction mixture to stabilize the enzyme dur-
4
ing incubations lasting longer than 16 h. Reactions were terminated by injection of
HClO (0.2 mL 4 M) through the septum to denature the protein and drive the
4
1
4
CO out of solution. To reduce chemiluminescence, the filter paper was incubated
2
overnight in ScintiVerse E scintillation fluid (Fisher Scientific) before scintillation
counting. In some experiments, the OMP decarboxylase inhibitor barbituric acid
0
ꢀ12
nucleoside 5 -phosphate (BMP, K = 8.8 · 10
M) [16] was used as a control.
i

48
A. Sievers, R. Wolfenden / Bioorganic Chemistry 33 (2005) 45–52
Catalytic rescue was attempted with NaNO , NaNO , Na HPO , and NaCl at a con-
3
2
2
3
centration of 100 mM.
2
.4. Assay of OMP decarboxylation
In control experiments, the decrease in activity of the enzyme during long incuba-
tions at room temperature was analyzed spectrophotometrically by monitoring the
ꢀ
1
1
ꢀ1
decrease in absorbance at 279 nm (De = 2.4 mM cm ) [15] due to conversion of
4
OMP (Sigma) to UMP. The decarboxylation of C-OMP was measured at a sub-
ꢀ7
strate concentration of 2.7 · 10 M, in Mops buffer (0.05 M, pH 7.2) containing
NaCl (0.1 M) and 10% glycerol.
3
. Results and discussion
A single phosphoryl group, 9 A away from the site of catalysis, distinguishes orot-
˚
idine from the normal substrate OMP. Since no enzymatic decarboxylation of orot-
idine could be detected by monitoring the UV absorbance change at 279 nm, a more
sensitive assay was needed to determine whether orotidine is a slow substrate. Fig. 2
1
4
shows that the amount of radioactivity released from [ C]orotidine increases line-
arly with time over 48 h in the presence of OMP decarboxylase at 21 ꢁC. The pro-
longed period of incubation required for these observations, even in the presence
of highly concentrated enzyme, rendered it desirable to test the stability of the en-
zyme. Enzyme activity was found to decrease approximately 25% over the course
of a 48 h incubation (Fig. 2), and a modest correction for this loss of activity was
made in estimating the enzymeÕs true activity on orotidine.
14
14
Fig. 2. The CO
2
released from [ C]orotidine increases linearly with time in the presence of ODCase (m).
release is observed with the catalytically inactive ODCase mutant D96A (d). The enzymatic
activity decreases by ꢁ25% over 48 h (·).
14
No CO
2

A. Sievers, R. Wolfenden / Bioorganic Chemistry 33 (2005) 45–52
49
1
4
The rate of [ C]orotidine decarboxylation was found to increase linearly with
increasing substrate concentration (Fig. 3). From the dependence of the rate on sub-
ꢀ
4
ꢀ1 ꢀ1
strate concentration, a value of kcat/K = 2 · 10
M
s
was calculated for the
m
decarboxylation of orotidine (Fig. 3). Compared with the decarboxylation of the
ꢀ7
ꢀ1 ꢀ1
normal substrate OMP (kcat/K = 3 · 10
M
s
), the absence of the phosphoryl
m
group decreases the second order rate constant for decarboxylation by a factor of
.5 · 10 . No significant rate enhancement was observed when nitrate, nitrite, phos-
phite or phosphate was added to the orotidine decarboxylation assay in an attempt
1
1
1
to ‘‘rescue’’ enzyme activity on the truncated substrate. The enzymeÕs activity on
ꢀ
12
orotidine was inhibited by the tight binding inhibitor BMP (K = 8.8 · 10
M)
i
[
16], indicating that the enzymeÕs low activity on orotidine is inherent in yeast
OMP decarboxylase, and does not arise from contamination with an unknown en-
zyme. Mutation of Asp-96 to alanine results in a protein that binds OMP with an
affinity comparable with the wild-type enzyme, but is devoid of catalytic activity
1
4
[
17]. When this inactive mutant enzyme was incubated with [ C]orotidine, no detect-
1
4
able CO was released over a period of 48 h (Fig. 2).
2
The very weak binding of orotidine, as indicated by the absence of any indication
ꢀ3
of substrate saturation at concentrations up to 9 · 10 M (Fig. 3), is confirmed by
the absence of any inhibition of the decarboxylation of OMP by orotidine, with
ꢀ
7
OMP present at a concentration (2 · 10 M) somewhat lower than its Km value
ꢀ
7
(
7 · 10 M). These latter observations, conducted at very low concentrations of en-
zyme and OMP, were subject to substantial experimental error because of the insta-
bility of yeast OMP decarboxylase at very high dilutions. These inhibition
ꢀ
2
experiments suggest a lower limit of K = 3 · 10 M for orotidine, consistent with
i
the lower limit suggested by the experiments using orotidine as a substrate (Fig. 3).
14
2
Fig. 3. The rate of CO release over 16 h increases linearly with the concentration of orotidine. No
saturation kinetics are observed up to a orotidine concentration of 9 mM. The rate of enzymatic activity is
corrected for the decrease in enzymatic activity over the time of incubation.

50
A. Sievers, R. Wolfenden / Bioorganic Chemistry 33 (2005) 45–52
The substantial error of this assay may account, at least in part, for the discrepancy
ꢀ
3
between the present findings and a K value of 1.5 · 10 M reported earlier for orot-
i
idine [18]. Because of solubility limitations, it was not possible to extend these obser-
ꢀ
2
vations to concentrations of orotidine higher than 2 · 10 M. Even if such
experiments had been feasible, it would have been difficult to interpret in view of
the possibility that very high concentrations of this nucleoside might affect activity
by changing the monomer–dimer equilibrium or substrate affinity [15].
In earlier work on triosephosphate isomerase, the presence of a single phosphoryl
group on the substrate has been shown to contribute 8 orders of magnitude to the
rate enhancement produced by triosephosphate isomerase, enough to account for al-
most all the rate enhancement produced by that enzyme [19]. In the present case, we
find that a single substrate phosphoryl group, by its presence, contributes 11 orders
of magnitude to the rate enhancement produced by ODCase. This contribution,
although it is the largest that appears to have been recorded for a simple substituent
1
7
in an enzyme reaction, does not fully account for the 10 -fold rate enhancement
produced by OMP decarboxylase. Comparing the K values of orotidine
m
ꢀ2
ꢀ6
(
>10 M) and OMP (10 M), it appears that at least 4 orders of magnitude of
the rate enhancement arise from binding interactions that do not discriminate be-
tween the substrate in the ground state and the transition state.
The maximal dissociation constant KTX of an altered substrate in the transition
state can be calculated by dividing the rate constant for the uncatalyzed reaction,
knon, by the second order rate constant k /K . For OMP, the value of K is
TX
cat
m
ꢀ
24
ꢀ13
4
.4 · 10
M [4], which may be compared with a value of 7 · 10
obtained in the present experiments. The dissociation constant of inorganic phos-
phate from yeast OMP decarboxylase is 7 · 10 M [9]. The sum of the negative free
energies of binding of the truncated substrate orotidine and the missing phosphate
falls 12.3 kcal short of the negative free energy of binding of the altered substrate
OMP in the transition state. This connectivity effect can be expressed as an effective
M for orotidine
ꢀ
3
9
concentration of 1.1 · 10 for the phosphoryl group in the transition state. Fig. 4
compares the connectivity effect in yeast OMP decarboxylase with connectivity
effects that have been reported for other enzymatic reactions.
The structural means by which these very large connectivity effects are expressed
in catalysis remains somewhat uncertain. The prospects of obtaining structural infor-
mation concerning the ability of yeast ODCase to discriminate between OMP and
orotidine appear to be clouded by this enzymeÕs weak affinity for orotidine. In the
reported structures of ODCase complexes with nucleotides, however, the phosphoryl
group exhibits multiple interactions with the enzyme (Gly-234, Arg-235, and Tyr-
2
17). It seems reasonable to speculate that the pronounced difference in transition
state binding affinity between these two similar substrates may be related to the
ordering and closing of loop 207–217 upon nucleotide binding [6]. In the ordered
form of this loop, there is a hydrogen bond between the hydroxyl group of Tyr-
2
17 and the phosphoryl group, and mutation of that residue to alanine reduces
the catalytic efficiency of yeast ODCase by 4.7 kcal/mol in free energy [9]. For such
a movement to lower the free energy of activation, the benefit of increased contact
would need to outweigh the cost of distorting the enzyme from its native, or open,

A. Sievers, R. Wolfenden / Bioorganic Chemistry 33 (2005) 45–52
51
Fig. 4. Connectivity effect in enzyme reactions. Many of the values in this figure [9,20] are dissociation
constants (KTX or K ), but the tabulated values for the two aldehydic substrates of triosephosphate
isomerase [19] describe kcat/K , which is inversely related to the value of the dissociation constant KTX
d
m
.

52
A. Sievers, R. Wolfenden / Bioorganic Chemistry 33 (2005) 45–52
structure. The cost of distorting the enzyme from its native open structure, which
must be paid as part of this process, would be expected to be minor if it involved
a hinge-like movement of two parts that were otherwise well ordered [21]. The con-
straints imposed by formation of one ‘‘weak’’ bond can enhance the probability of
forming a second, and such effects may in principle raise the apparent concentration
of the reaction partner to high levels (3). In earlier work, effective concentrations of
5
8
1
0 –10 M have been determined experimentally by making ‘‘cuts’’ in enzymes or
substrates, then comparing the binding affinities of these ‘‘pieces’’ with those of
the native enzyme or substrate in the transition state [22]. Combined with the large
binding contributions that have been observed for individual groups [23], these levels
of synergism appear to bring the transition state affinities of enzymes within reach of
ordinary forces of attraction.
Acknowledgments
This work was funded by NIH Grant GM-18325 (R.W.), and NIH Training
Grant GM-08570 (A.S.).
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