A substantial oxygen isotope effect at O2 in the OMP decarboxylase reaction: Mechanistic implications

Article abstract of DOI:10.1039/b812979g

Orotidine-5′-monophosphate decarboxylase (OMP decarboxylase, ODCase) catalyzes the decarboxylation of orotidine-5′-monophosphate (OMP) to uridine-5′-monophosphate (UMP). Despite extensive enzymological, structural, and computational studies, the mechanism of ODCase remains incompletely characterized. Herein, carbon kinetic isotope effects were measured for both the natural abundance substrate and a substrate mixture synthesized for the purpose of carrying out the remote double label isotope effect procedure, with O2 of the substrate as the remote position. The carbon kinetic isotope effect on enzymatic decarboxylation of this substrate mix was measured to be 1.0199 ± 0.0007, compared to the value of 1.0289 ± 0.0009 for natural abundance OMP, revealing an ¹⁸O2 isotope effect of 0.991 ± 0.001. This value equates to an intrinsic isotope effect of approximately 0.983, using a calculated commitment factor derived from previous isotope effect data. The measured ¹⁸O2 isotope effect requires a mechanism with one or more enzymatic processes, including binding and/or chemistry, that contribute to this substantial inverse isotope effect. ¹⁸O2 kinetic isotope effects were calculated for four proposed mechanisms: decarboxylation preceded by proton transfer to 1) O2; 2) O4; and 3) C5; and 4) decarboxylation without a preceding protonation step. A mechanism involving no pre-decarboxylation step does not appear to have any steps with the necessary substantial inverse ¹⁸O2 effect, thus calling into question any mechanism involving simple direct decarboxylation. Protonation at O2, O4, or C5 are all calculated to proceed with inverse ¹⁸O2 effects, and could contribute to the experimentally measured value. Recent crystal structures indicate that O2 of the substrate appears to be involved in an intricate bonding arrangement involving the substrate phosphoryl group, an enzyme Gln side chain, and a bound water molecule; this interaction likely contributes to the observed isotope effect.

Full text of DOI:10.1039/b812979g

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Volume 6 | Number 24 | 21 December 2008 | Pages 4469–4672
ISSN 1477-0520
FULL PAPER
2
WT\XRP[ ꢀB RXT]RT
Wickliffe O. Wepukhulu et al.
A substantial oxygen isotope effect at
O2 in the OMP decarboxylase reaction:
Mechanistic implications
In this issue...
1477-0520(2008)6:24;1-G

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A substantial oxygen isotope effect at O2 in the OMP decarboxylase
reaction: Mechanistic implications†
a
a
a
a
Wickliffe O. Wepukhulu,‡ Vanessa L. Smiley,§ Bhargavi Vemulapalli,¶ Jeffrey A. Smiley,*ꢀ
b
b
Linda M. Phillips and Jeehiun K. Lee*
Received 28th July 2008, Accepted 24th September 2008
First published as an Advance Article on the web 30th October 2008
DOI: 10.1039/b812979g
Orotidine-5¢-monophosphate decarboxylase (OMP decarboxylase, ODCase) catalyzes the
decarboxylation of orotidine-5¢-monophosphate (OMP) to uridine-5¢-monophosphate (UMP). Despite
extensive enzymological, structural, and computational studies, the mechanism of ODCase remains
incompletely characterized. Herein, carbon kinetic isotope effects were measured for both the natural
abundance substrate and a substrate mixture synthesized for the purpose of carrying out the remote
double label isotope effect procedure, with O2 of the substrate as the remote position. The carbon
kinetic isotope effect on enzymatic decarboxylation of this substrate mix was measured to be 1.0199 ±
1
8
0
.0007, compared to the value of 1.0289 ± 0.0009 for natural abundance OMP, revealing an O2
isotope effect of 0.991 ± 0.001. This value equates to an intrinsic isotope effect of approximately 0.983,
1
8
using a calculated commitment factor derived from previous isotope effect data. The measured O2
isotope effect requires a mechanism with one or more enzymatic processes, including binding and/or
1
8
chemistry, that contribute to this substantial inverse isotope effect. O2 kinetic isotope effects were
calculated for four proposed mechanisms: decarboxylation preceded by proton transfer to 1) O2; 2) O4;
and 3) C5; and 4) decarboxylation without a preceding protonation step. A mechanism involving no
1
8
pre-decarboxylation step does not appear to have any steps with the necessary substantial inverse O2
effect, thus calling into question any mechanism involving simple direct decarboxylation. Protonation
1
8
at O2, O4, or C5 are all calculated to proceed with inverse O2 effects, and could contribute to the
experimentally measured value. Recent crystal structures indicate that O2 of the substrate appears to be
involved in an intricate bonding arrangement involving the substrate phosphoryl group, an enzyme Gln
side chain, and a bound water molecule; this interaction likely contributes to the observed isotope effect.
Introduction
and only surpassed since then by enzymes utilizing cofactors. The
cofactor-free ODCase from four microbial sources yielded to crys-
Orotidine-5¢-monophosphate decarboxylase (OMP decarboxy-
lase, ODCase; E.C. 4.1.1.23) catalyzes the decarboxyla-
tion of orotidine-5¢-monophosphate (OMP) to uridine-5¢-
monophosphate (UMP, eqn 1) and is a key enzyme in the de
novo pathway of pyrimidine biosynthesis. ODCase has received a
degree of attention in the scientific press that is unusual for most
enzymes. This heightened attention became especially pronounced
upon the observation that ODCase displays a catalytic rate
tallographic determination in nearly simultaneous publications in
4–7
2
000. Since those structural studies, the attempts at acquiring
mechanistic information using enzymological, computational,
2,8
and further structural studies have been numerous. Nonetheless,
the mechanism of ODCase remains incompletely characterized.
1,2
3
1
7
enhancement, kcat/kuncat, of >10 , a factor then unprecedented,
a
Department of Chemistry, Youngstown State University, Youngstown, OH
4555, USA. E-mail: j_smiley@acs.org; Tel: +1 (202) 872-6093; Fax: +1
202) 872-6319
4
(
b
Department of Chemistry and Chemical Biology, Rutgers University,
Piscataway, NJ 08854, USA. E-mail: jeehiun@rci.rutgers.edu; Tel: +1
(1)
(
732) 445-6562; Fax: +1 (732) 445-5312
†
Electronic supplementary information (ESI) available: Mass spectromet-
ric data indicating isotope enrichment specifically at the O2 position in
uracil, and the double enrichment in [ O, C-carboxyl] OMP. Cartesian
coordinates for all calculated species are also available.
Several mechanisms for the ODCase-catalyzed reaction have
been proposed (Fig. 1). A long-standing mechanistic proposal
first advanced by Beak and Siegel arose from the observations
of different rates of decarboxylation for model compounds,
and involves protonation at O2 of the substrate (Fig. 1A). A
second mechanism involving protonation of O4 followed by
decarboxylation to yield a stabilized carbene was put forth by Lee
18
13
9
‡
§
¶
Current address: Merck and Co., Inc., West Point, PA, USA.
Current address: Panacea Pharmaceuticals, Gaithersburg, MD, USA.
Current address: MedImmune, Gaithersburg, MD, USA.
Permanent address: American Chemical Society, Petroleum Research
ꢀ
Fund, 1155 Sixteenth St. NW, Washington, DC 20036, USA.
This journal is © The Royal Society of Chemistry 2008
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Fig. 1 Four possible mechanisms of ODCase-catalyzed OMP decarboxylation. R = 5-phospho-1-ribosyl (see eqn 1). Protonated intermediates (reactions
A–C) are not expected to have isolated positive charges; they are shown as single resonance species to highlight the difference in protonation sites.
1
0
and Houk (Fig. 1B). Protonation at C5 has also been proposed
(
protonation, especially protonation at O2 (Fig. 1A). A small,
normal N1 isotope effect of 1.0036 was measured for the OD-
Case reaction using the yeast enzyme; an intrinsic N1 isotope
effect of 1.0068 was calculated. The interpretation included an
assumption of the equilibrium isotope effect (EIE) for N1 in
the O2 protonation mechanism to be comparable to the EIE
for N protonation of pyridine, a value that had been previously
measured to be 0.979. The use of this substantial inverse EIE in the
interpretation appeared to rule out an O2 protonation mechanism,
with the reasoning that the O2 protonation step would necessarily
contribute to a substantial inverse isotope effect for the measured
11
Fig. 1C).
A fourth possibility is direct decarboxylation (Fig. 1D), which
does not involve a pre-decarboxylation protonation.
mechanism involves no modifications of the pyrimidine ring, but
simply a destabilizing interaction of the substrate carboxylate
with the catalytic tetrad Lys44-Asp71-Lys73-Asp76(B) (amino
acid numbering from the E. coli sequence). The mechanism was
predicated on computational studies, even though other com-
putational studies came to different conclusions.
in direct decarboxylation also exist: some involve generation of
a decarboxylated carbanion
a product deuterium isotope effect study and observation of
evidence for an anionic intermediate essentially eliminated the
possibility of a direct decarboxylation mechanism with no inter-
mediate. The controversial topic of ground state destabilization
has been invoked at times to accompany the direct decarboxylation
mechanism, and subsequently challenged.
Some mechanistic studies indicate the presence of a proton-
dependent step prior to decarboxylation in the reaction. The
pattern of isotope effects at varying pH suggests a kinetically
discernable pH-dependent step for the yeast enzyme, a mechanistic
possibility that was supported by the determination of carbon
4,6,7,12
This
13, 14
Variations
6,12
4,7
21
while others do not. Recently,
reaction. A subsequent theoretical analysis, however, indicated
15
a very small N1 EIE of 0.997 upon O2 protonation, much less
substantial than that for the EIE for N protonation in pyridine.
The bond order of N1 does not change appreciably upon O2
protonation, and thus a small, normal N1 isotope effect would be
expected for a mechanism involving OMP protonation at O2. The
nitrogen isotope effect data does not appear to distinguish between
a mechanism with a small N1 EIE (such as the O2 protonation
16
4,6
17
mechanism) and an N1-independent mechanism.
Very recently, new crystal structures have been presented
18
22,23
that
clarify further the binding and decarboxylation of the substrate
22
OMP. The Inoue and Krungkrai groups have achieved the
crystallization of the wild type Plasmodium falciparum enzyme
in the presence of the substrate. Apparently, the complexation of
the active enzyme with substrate did not proceed on to decar-
boxylation, possibly due to the inability of the crystallized enzyme
to undergo a conformational change necessary for catalysis at the
low temperatures used. In the P. falciparum structure, the substrate
binds in an orientation similar to that for the inhibitors seen in
previous crystal structures, with the carboxylate in proximity of
the catalytic tetrad. The structure of the human ODCase domain
isotope effects in H
2
O and D O with the ODCase from Escherichia
2
19
coli. Despite this evidence for a pH-dependent step, protonation
of the orotate ring to yield a kinetically discernable intermediate
prior to decarboxylation was omitted in the formulation of
the direct decarboxylation mechanisms, presumably with the
assumption that the pH-dependent step was related to protonation
elsewhere within the enzyme or substrate.
20
An examination of nitrogen isotope effects at N1 of OMP
has been cited as evidence against mechanisms involving ring
4
534 | Org. Biomol. Chem., 2008, 6, 4533–4541
This journal is © The Royal Society of Chemistry 2008

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2
3
of UMP synthase by the Rudolph group shows the substrate in
complex with a highly weakened D312N mutant (corresponding
to Asp71 of the E. coli sequence); this is a minimal perturbation
that essentially maintains the active site volume available for
OMP binding. The OMP structure shows a dramatic out-of-plane
bending of the labile carboxylate.
Table 1 Carbon kinetic isotope effects using OMP synthesized for the
a
remote double label method
13
13
d
C
f
k
Natural abundance OMP
-
-
-
-
72.70
52.46
72.65
52.46
0.389
1.0
0.459
1.0
0.326
1.0
0.48
1.0
1.0282
1.0300
1.0278
1.0295
However, O2 of the substrate is still seemingly involved in a
critical feature of the structure: in the human D312N structure,
which may resemble a snapshot of the decarboxylation reaction in
progress, the substrate’s phosphoryl group closely approaches O2.
In fact, one O atom of the phosphoryl group is almost co-linear
with the C2-O2 carbonyl group. The two aligned oxygen atoms are
bridged, at an angle, by the amide N of Gln430; analogous Gln
residues are present in all structures. A bound water molecule is
also present in another bridging position between the phosphoryl
group and O2 (ref. 23, PDB ID# 2QCL). These features of the
structure suggest an intricate network of contacts at O2 that
-71.88
-
-
-
50.80
69.70
50.25
13
k = 1.0289 ± 0.0009
b
Remote label OMP mixture
-
34.12
17.15
0.30
1.0
0.595
1.0
0.44
1.0
0.37
1.0
1.0211
1.0198
1.0193
1.0192
-
-25.64
-
-
-
-
13.74
52.85
39.33
53.61
24
appear important for the reaction. The Gerlt and Richard groups
explored this portion of the active site through mutagenesis of the
conserved Gln residue and a neighboring Ser residue of the E. coli
enzyme.
In this paper, we approach the ODCase reaction using oxygen
isotope effects in a combined experimental-theoretical study
designed to probe the role of O2 of OMP in the ODCase reaction.
-39.33
1
3
8
k = 1.0199 ± 0.0007
1
O effect = 0.991 ± 0.001
a
13
f : fraction of reaction. k: carbon kinetic isotope effect measurement,
13
calculated from the two measured d C values and the corresponding f
values for the partial reaction. “Remote Label OMP Mixture” refers to
b
1
8
Experimentally, we utilized O enrichment and the double label
18
13
the substrate mixture described in the text: a mixture of [ O2, C-carboxyl]
OMP, and OMP depleted in both positions. The ratio of the two isotope
effects is equal to the O effect on the carbon kinetic isotope effect.
25
method described by O’Leary. In this approach, the carbon
isotope effect (at the carboxylate carbon) is measured for the
enzymatic reaction using the natural abundance substrate. Then,
a remote label substrate mixture is synthesized: in the ODCase
system, isotopically doubly-enriched OMP contains O at O2 and
C in the carboxylate carbon. Separately, OMP is synthesized with
heavy atom depletion at both positions. The natural abundance of
O is sufficiently low as to be considered isotopically depleted; in
this study, the depleted OMP was synthesized with C-depleted
carbon at the carboxylate position. The isotopically enriched and
depleted substrates are then combined so that the C (from the
enriched OMP) is near natural abundance, requiring a ratio of
approximately 1:90 of the doubly-enriched to doubly-depleted
OMP. The carbon isotope effect for the enzymatic reaction using
this mixture is then measured; the ratio of the two carbon isotope
effects is equal to the isotope effect at O2 for the enzymatic reac-
tion. We measured carbon isotope effects for natural abundance
OMP and an OMP remote label mixture in order to probe the
possible reaction mechanisms for the ODCase reaction. Isotope
effects were calculated for several possible mechanistic steps and
compared to the experimental result in order to determine which
mechanisms could be supported.
18
was necessary to preserve isotopic enrichment throughout the
1
8
18
13
synthesis of OMP. The complete synthesis of [ O2, C-carboxyl]
OMP resulted in high isotopic enrichment at both positions, indi-
cated from the predominant anion MW = 370 versus anion MW =
367 for natural abundance OMP (see Supplementary Materials).
A sample of 10 mmol of isotopically depleted OMP was
1
3
1
8
1
3
decarboxylated with ODCase, in order to assess the level of
1
3
13
C depletion. The CO
2
collected showed a delta value of -370
from natural abundance OMP ª -50),
(cf., delta value of CO
indicating extensive C depletion.
2
1
3
The carbon kinetic isotope effect for E. coli ODCase measured
in this study, for the natural abundance substrate, was 1.0289 ±
◦
0.0009, at pH 7.0, 25 C (Table 1). This can be compared with
19
the previously reported value of 1.043 ± 0.003, at pH 7.5. E. coli
ODCase thus displays a sharp increase in the kinetic isotope effect
of decarboxylation as the pH is raised above neutral, a trait that
18
was also described for the yeast ODCase.
The carbon kinetic isotope effect for ODCase with the remote
label substrate mixture, to assess the isotope effect at O2, was
1.0199 ± 0.0007 (Table 1). This isotope effect was essentially
independent of composition of the remote label substrate mix;
different mixes gave the same result. The ratio of the two kinetic
isotope effects, 0.991 ± 0.001, indicates the isotope effect at O2 for
the overall reaction.
Since the enrichment and depletion of isotopes in the remote
label OMP mixture are not complete, a small correction to the
measured value can be made using the equations described by
Results and discussion
Measurement of the isotope effects
1
8
Uracil synthesized by the method described, with H
ing the 2-ethylthio group, showed a molecular weight of 114
anion = 113), with very little MW = 112 and MW = 116 (see Sup-
plementary Materials), indicating nearly exclusive incorporation
2
O hydrolyz-
(
2
6
18
18
Cleland. Using 97% enrichment for O (from the O water used
1
8
18
13
13
-
of O into only the 2-position. This O-enriched compound was
in the synthesis), 99% enrichment for C (from the CN used) and
0.2% natural abundance O in the depleted OMP, the measured
effect of 0.991 corrects to 0.990. Using 98.5% enrichment for
1
8
stable to 0.5 M NaOH, with no observable conversion of MW =
1
8
1
14 to MW = 112 (see Supplementary Materials). This stability
O
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1
3
and 99.5% enrichment for C in the calculation also results in the
correction to 0.990.
motion is an inverse effect in the range of 0.994. It seems reasonable
to assume that torsional motions at O2 of OMP will not be as
significantly affected as is the case for the bridging oxygen in PEP:
both the C–O and O–P bonds are freely rotatable in PEP, while O2
of OMP does not have the same role in conformational flexibility
as the bridging oxygen of PEP.
Measurement of the ODCase isotope effect under neutral
conditions does not allow observation of any intrinsic isotope
effect, due to the presence of a substantial commitment factor.
The largest C carboxylate KIE measured for ODCase, 1.0506,
is likely very close to the intrinsic C carboxylate IE. Using this
value, and the C carboxylate KIE of 1.0289 under conditions
used herein, a commitment factor of 0.75 can be calculated.
Applying this commitment factor to the O2 effect yields an
intrinsic O2 isotope effect of 0.983. The error in this value
is difficult to predict, due to the uncertainty of the intrinsic
C carboxylate IE and uncertainty about whether or not the
18,20
1
3
1
3
These two effects possibly contributing to Step A are therefore
small and opposite to each other, and taken together likely
contribute no more than 0.003 in either direction to the overall
1
3
1
8
18
ODCase O IE.
1
8
18
O2 isotope effects for decarboxylation steps were calculated for
the three proposed protonated intermediates as well as for unpro-
1
3
18
tonated OMP. In every case, the O IE was calculated to be small
1
3
commitment factor for the C-carboxyl position is identical to
the commitment factor for O2, so this value will be considered
approximate in the ensuing discussion.
and normal; a range of 1.003 to 1.006 was found (Fig. 2). The direc-
tion of this isotope effect is predictable, since decarboxylation re-
sults in increased electron density on the ring and thus a longer C–
O2 bond in the transition state. Such a reaction would be slightly
1
8
Interpretation of the O2 isotope effect
1
6
18
faster for O than for O, giving rise to a normal isotope effect.
For the direct decarboxylation mechanism to be congruent with
the observed data, it is apparent that a large contribution to the
The mechanism for ODCase decarboxylation of OMP must
include binding and chemical steps which, taken together, account
1
8
18
for the substantial O2 isotope effect of 0.983. In a multi-step
enzymatic reaction, small isotope effects may arise at almost any
step. The overall IE will be the product of IEs on all steps up to and
including the first irreversible step in the reaction; for the ODCase
reaction under our conditions, the decarboxylation step can be
assumed to be irreversible. Previous isotope effects measured in
O and D O evidenced the presence of a pH-dependent pre-
decarboxylation step. In consideration of a minimal kinetic
mechanism, where ES† is an activated intermediate (eqn 2):
O2 isotope effect of 0.983 present in the ODCase reaction would
have to come from Step A. The assumption that Step B does not
involve chemistry on the orotate ring would require that that step
is isotopically insensitive, or essentially insensitive, at O2. Since
the decarboxylation step (C) is expected to contribute a normal
isotope effect of 1.004–1.005, step A must contribute a factor in
the range of 0.980–0.985 to the observed effect.
H
2
2
19
In an attempt to gain information on the isotope effect at O2
upon binding, we calculated the equilibrium IE associated with
simple hydrogen bond formation between O2 of 1-methylorotate
and the NH of formamide, in a geometry similar to that seen in
the ODCase crystal structures (Fig. 3). The effect of hydrogen
bonding was explored by calculating the isotope effect associated
with the complexation of 1-methyl orotate with acetamide (which
mimics glutamine; details in Computational methods section). The
isotope effect associated with acetamide forming two hydrogen
bonds as shown in Fig. 3 is 0.9998. Therefore, this routine
hydrogen bonding and a pre-decarboxylation step not involving
bond-making on the orotate ring in the direct decarboxylation
A
B
†
C
E + S ꢂꢀ ꢀꢀ ꢀꢁꢀ ES ꢂꢀ ꢀꢀ ꢁꢀꢀ ES æ æÆ E + P
(2)
all possible ODCase mechanisms must involve substrate binding
A) and decarboxylation (C).
The various protonation mechanisms propose formation of ki-
(
netically discernable protonated intermediates (Fig. 1) that would
account for Step B in eqn 2. Step B in the direct decarboxylation
mechanism must be a chemical or conformation step that does not
involve chemistry at the orotate ring.
An isotope effect on Step A can arise from a number of factors
not involving direct contact of the enzyme with the isotopic
position, as recently reviewed by Schramm. In the case of
ODCase, at least two factors are possibly involved: desolvation
of the substrate, and restricted molecular motion upon binding.
Desolvation of O2 of OMP would likely contribute a normal
1
8
mechanism cannot account for the measured substantial O2
effect if the effects for the proposed individual steps are consistent
with our modeled values.
27
1
8
In a further attempt to interpret the O2 isotope effect,
we calculated equilibrium IEs associated with the individual
protonation steps for the mechanisms in Fig. 1, using N1-methyl
1
8
21
(
>1) component to the O effect for the overall reaction. The
orotate as the substrate. The calculated EIEs for the equilibrium
desolvation of formate in the formate dehydrogenase reaction was
in the gas phase are shown in Fig. 2, above the equilibrium arrows.
For the O2 protonation mechanism (Fig. 2A), the initial proton
1
8
predicted to have a small, normal contribution to the overall
O
28
18
IE. Transfer of water into the vapor phase is accompanied by an
transfer is associated with an inverse O2 EIE of 0.993.
1
8
29
18
O IE of 1.009. O2 of OMP would seem to be less highly solvated
Intriguingly, the O2 EIE for the O4 protonation (Fig. 2B) is
than formate (charged) or water molecules, so the desolvation
contribution to the isotope effect in the case of the ODCase
reaction would likely be minimal.
similar, 0.994. This is somewhat surprising since one might expect a
much more substantial inverse IE for protonation at O2 than at O4,
1
8
since the O substitution is at O2. However, these isotope effects
arise not only from new bond formation at enriched positions
(as for the O2 protonation mechanism) but also from changes in
geometry, and O4 protonation incurs a shortening of the C–O2
bond, leading to the inverse IE. Protonation of O2 results in a new
bond at the labeled position, which would contribute to an inverse
IE, but the C–O2 bond becomes longer, contributing a normal
Restricted torsional motions of a substrate upon binding to an
enzyme active site are also known to contribute to an IE for an
1
8
enzyme reaction. A thoroughly discussed example of O effects is
given for two enzyme reactions that utilize phosphoenolpyruvate
3
0
(
PEP), examining the effects at the C–O–P bridging oxygen. The
1
8
O effect at this position upon binding and restriction of torsional
4
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Fig. 2 Calculated isotope effects for possible ODCase mechanisms. Gas phase values for the protonation equilibria are above the equilibrium arrows;
values in a water dielectric are below the arrows.
1
8
interesting literature precedent for O isotope effects on binding
is found in the study of lactate dehydrogenase (LADH) and its
1
8
31
18
interaction with [1- O] oxamate, a substrate analog. The
O
effect on binding was measured to be 0.984 ± 0.003; this effect is
attributable to an intricate interaction of the labeled carboxylate
group of oxamate with two guanidinium groups from arginine side
chains and a threonine hydroxyl group. In the LADH-oxamate
32
structure, either oxygen in the carboxylate group will be involved
in two hydrogen bonding or charge interactions with functional
groups at the active site, regardless of the orientation of the labeled
oxygen in the carboxylate. An isotope effect of this magnitude in
the ODCase-OMP interaction would almost completely account
for the approximate observed value of 0.983, with small effects
from the other steps factored in.
Fig. 3 Calculated optimized complex of acetamide and 1-methyl orotate,
at B3LYP/6-31 + G*.
component to the IE and resulting in a protonation EIE value
that is similar to that of protonation at O4. The C5 protonation
mechanism (Fig. 2C) has a calculated protonation EIE of 0.992.
In an attempt to model a more enzyme-like environment, we also
calculated the EIE for the protonation step in a water dielectric.
We find that the values do not drastically change when solvent is
taken into account (Fig. 2, water values below equilibrium arrows).
The O2 protonation is slightly more inverse in water than in the
gas phase, but by very little (0.993 gas phase, 0.992 in water).
The O4 protonation shows the greatest change, from an EIE of
22,23
The orientation of O2 in the new ODCase-OMP structures
indicates a significant interaction of O2 with the substrate
phosphoryl group, an enzyme Gln side chain, and a bound
water molecule. This interaction, which may involve proton
transfer or hydrogen bonding, appears to offer an explanation
for the observed isotope effect data. Considering the enormous
(11 orders of magnitude) decrease in ODCase activity toward
33
0
.994 in the gas phase to 0.996 in water. The C5 protonation
mechanism remains essentially constant (0.992 in the gas phase,
.993 in water). The direct decarboxylation mechanism is also
orotidine nucleosides lacking phosphoryl groups and the rescue
34
of activity using phosphite, and the very large (at least 7 orders
35
0
of magnitude) decrease in ODCase activity toward 2-thioOMP,
barely affected by solvation and stays firmly normal (IE of 1.004
in gas phase, 1.005 in water). In essence, the general trends remain
unchanged: the protonation steps are characterized by an inverse
O2 IE, and decarboxylation has a normal O2 IE.
These calculated EIE values for protonation are somewhat
this proposed feature of the enzyme reaction, which still remains
incompletely characterized, seems critical to enzyme activity.
Although the collection of functional groups in the proximity
of O2 in the ODCase-OMP structure appears more intricate than
that at O4 or C5, there does not appear to be any firm experimental
data yet presented that excludes the proposed mechanisms of
protonation at these sites. It should be noted that most depictions
1
8
18
small, and another feature of the reaction is likely necessary
1
8
to fully account for the O2 effect of approximately 0.983. An
This journal is © The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 4533–4541 | 4537

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of ODCase active sites omit the presence of enzyme-bound
water molecules, which have been proposed to be proton transfer
conduits.
Standard HPLC conditions for analysis of nucleotides
Using a Waters 1525 dual pump HPLC (Breeze System software),
36
a gradient of water as solvent A and 0.8 M NH HCO as solvent
B was run at a flow rate of 2 mL min with the following setup:
min, 100% A, 0% B; 20 min, 0% A, 100% B. The column was a
4
3
-
1
0
Experimental
PRP-X100 anion exchange column (250 mm ¥ 4.6 mm, Hamilton).
Elution was analyzed spectrophotometrically at 280 nm, except in
the case of the monitoring of the fraction of conversion of OMP
to UMP (see below), in which case the wavelength was 264 nm.
Injections of standard nucleotide solutions (100 nmol nucleotide)
were performed to yield peaks for general retention times of each
nucleotide: UMP (6.4 min), 5-Br-UMP (9.3 min), 6-CN-UMP
Production of E. coli ODCase
37
The ODCase gene from E. coli was amplified using PCR, for
insertion into the NdeI and HindIII restriction sites of plasmid
pCAL-n (Stratagene). Oligonucleotide primers for PCR were
obtained from Integrated DNA Technologies, Inc., and have the
following sequences (restriction sites underlined):
(
11.0 min), OMP (7.2 min).
5
¢ primer: 5¢-GGGAAAGGCATATGACGTTAACTGCTTC-
¯
¯ ¯ ¯ ¯ ¯
ATCTTC-3¢
Standard ion exchange chromatography conditions for purification
of nucleotides
3
¢ primer: 5¢-GGAAAGGAAAGCTTTCATGCACTCCGC-
¯
¯ ¯ ¯ ¯ ¯
TGTAAAGAGG-3¢
Thermostable DNA polymerase from New England Biolabs
Nucleotide solutions were applied to a column (2.5 cm diameter
was used in a standard PCR mixture with the above primers and a
and lengths from 20 to 28 cm) filled with Dowex 1 ¥ 8–200 anion ex-
38
preparation of E. coli genomic DNA. The resulting 0.8 kbp PCR
product was purified, digested with NdeI and HindIII (NEB),
purified again and ligated to pCAL-n digested with the same
restriction enzymes, using T4 DNA ligase (NEB). The ligation mix
was used for transformation of E. coli XLI-Blue to give ampicillin
resistance, and potentially correct plasmid constructions from
transformants were screened by gel electrophoresis of plasmid
preparations. Plasmids appearing to contain the ODCase gene
were introduced into E. coli BL21 (DE3)-Gold cells (Stratagene).
Protein lysates from cultures carrying this plasmid and induced
change resin (Sigma/Aldrich) washed with 0.8 M NH
rinsed with dH O. A gradient from 0 to 0.8 M NH HCO
each) was used for elution, with additional 0.8 M NH HCO
4
HCO
(0.5 L of
used
3
and
2
4
3
4
3
for continued elution as necessary. Fractions (8 mL) containing
nucleotide were identified by UV absorbance and subsequently
analyzed by HPLC (as described above). Fractions containing the
desirednucleotide were pooledanddesaltedwithDowexHCR-W2
cation exchange resin (Sigma/Aldrich), which was washed prior
to desalting with 1 M HCl and then rinsed with dH O until the
2
rinsings were neutral. Enough resin was added to the nucleotide
solution so that the pH was lowered to less than 4. Resin was
removed by filtration and solutions were evaporated to a desired
concentration, or dryness. Final concentrations were determined
spectrophotometrically at absorbances appropriate to the specific
◦
with IPTG (50 mM; incubation continued at 37 C for 6 hrs after
IPTG addition to mid-log culture in LB media) showed vastly
39
increased levels of ODCase activity versus uninduced cells, based
40
on ODCase spectrophotometric assays.
ODCase was purified using Affi-Gel Blue (BioRad) chromatog-
42
nucleotide.
41
raphy, using a method similar to that previously described,
except that ODCase could be eluted from the column using
mM UMP instead of the stronger inhibitor 6-azaUMP used
previously. Dialysis of the pooled, concentrated fractions resulted
18
13
Synthesis of [ O2, C-carboxyl] OMP
1
2-Thiouracil was converted to 2-ethylthiouracil (Fig. 4, Step 1) via
39
in a decrease of UMP concentration to a very low level, as
analyzed by HPLC (see HPLC section below). ODCase prepared
by this method appeared as a single predominant band on gel
a reaction of 1.0 g of 2-thiouracil (Aldrich) with 1.2 g iodoethane
◦
in 15 mL DMF, with heating at 60 C under reflux. The product
was purified by silica gel chromatography using a solvent of
2:3 hexane/ethyl acetate.
-
1
-1 39
electrophoresis, with specific activity of 55 nmol min mg .
18
13
Fig. 4 Synthetic scheme for [ O2, C-carboxyl] OMP. Details are described in the text. Step 1: Alkylation of 2-thiouracil with iodoethane. Step 2:
1
8
18
18
2
Hydrolysis of 2-ethylthiouracil with acidified H O. Step 3: Conversion of [ O2] uracil to [ O2] UMP with UPRTase and PRPP. Step 4: Conversion of
18
18
18
18
13
[
2
O2] UMP to [ O2] 5-bromo-UMP using Br . Step 5: Conversion of [ O2] 5-bromo-UMP to [ O2, C-cyano] 6-cyano-UMP using NaCN or KCN. Step
: Hydrolysis of [ O2, C-cyano] 6-cyano-UMP to [ O2, C-carboxyl] OMP using aqueous NaOH. Steps 4–6 were used for the synthesis of natural
18
13
18
13
6
abundance and isotopically-depleted OMP, using natural abundance or isotopically-depleted reagents. R–P = 5-phospho-1-ribosyl.
4
538 | Org. Biomol. Chem., 2008, 6, 4533–4541
This journal is © The Royal Society of Chemistry 2008

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1
8
13
2
-Ethylthiouracil was converted to [ O2] uracil (Fig. 4, Step 2)
C-carboxyl]-OMP was analyzed by LC-MS, as above, to ensure
1
8
18
13
by acid-catalyzed hydrolysis in acidified H
2
O. HCl gas was bub-
retention of O and C enrichment.
1
8
18
bled through H
2
O (> 97% O, Cambridge Isotope Laboratories)
until the resulting solution was thoroughly acidic. 200 mg of 2-
Synthesis of natural abundance OMP
1
8
ethylthiouracil was added to 1 mL of acidified H O; the reaction
2
Conversion of UMP (Sigma-Aldrich) to OMP by the method
vial was sealed, vented with a hypodermic needle, and heated at
42
◦
of Ueda et al. was used with the following modifications: 1)
Fig. 4, Step 4) 5-Br-UMP was purified from its reaction solution
by evaporation to dryness and application of the redissolved
aqueous) solution to the standard ion exchange chromatography
7
0 C. Evolution of gas (thioethane) was observed, and the heating
(
continued until gas evolution ceased. Samples of the reaction mix
were analyzed by TLC (1:19 methanol/ethyl acetate solvent) with
(
2
-ethylthiouracil and uracil standards; heating was resumed until
conditions (above). 2) (Fig. 4, Step 5) 6-CN-UMP formation
was monitored using HPLC (above conditions) and the desired
product was again purified using the standard ion exchange
chromatography conditions (above). 3) (Fig. 4, Step 6) Conversion
of 6-CN-UMP to OMP was found to proceed to completion, with
formation of essentially no detectable by-products, using 0.5 M
NaOH at room temperature for 4 days. The OMP solution was
neutralized with Dowex HCR-W2 cation exchange resin, as above,
and used without further purification.
no more 2-ethylthiouracil could be observed in the reaction mix.
The resulting uracil was analyzed using a Bruker Esquire mass
spectrometer, under electrospray ionization conditions for anion
detection.
1
8
To determine whether the pyrimidine O2 was stable to base
1
8
hydrolysis conditions, in anticipation of the conversion of [ O2,
1
3
18
6
- C-cyano] 6-CN-UMP to OMP, approximately 1 mg of [ O2]-
uracil was placed in solutions containing a final concentration
of 0.5 M NaOH and held at room temperature for up to
8
days. Samples were periodically taken and analyzed by LC-
1
3
Synthesis of OMP ( C-depleted in the carboxylate)
MS to observe whether an exchange of natural abundance oxygen
from water could be detected, as indicated by conversion of the
molecular weight of 114 (for [ O2]-uracil) to 112 (for natural
abundance).
This synthesis was carried out as described above for synthesis of
natural abundance and enriched OMP, except that C-depleted
KCN (> 99.9% C, Icon Isotopes, Summit, NJ) was substituted
for natural abundance NaCN.
1
8
13
12
1
8
18
[
O2] uracil was converted to [ O2] UMP (Fig. 4, Step 3) using
recombinant UPRTase and the co-substrate PRPP (Fluka). The
43
UPRTase gene from genomic E. coli DNA was amplified by PCR
in a manner similar to the cloning of the E. coli ODCase gene, with
the following primers (IDT, Inc.) used:
Measurement of carbon isotope effects
25
General procedures were taken from O’Leary. CO samples were
2
5
¢ primer: 5¢-AGGTACATATGAAGATCGTGGAAGTCA-
analyzed for carbon isotope content using an Isoprime Dual Inlet
isotope ratio mass spectrometer (GV Instruments, Inc.). Reference
¯
¯ ¯ ¯ ¯ ¯
AA-3¢
3
¢ primer: 5¢-AGGTAAAGCTTTTATTTCGTACCAAAGAT-
gas was natural abundance CO (Praxair), which was analyzed
2
¯
¯ ¯ ¯ ¯ ¯
13
TTT-3¢
to have a C delta value of 0.21 versus Vienna PDB at the
Cornell University Stable Isotope Laboratory (Art Kasson, Lab
Manager).
Plasmids appearing to contain the UPRTase gene were intro-
duced into BL21 (DE3)-Gold cells. Protein lysates from cultures
induced with IPTG (as above for ODCase production) showed
vastly increased levels of a protein of 22.5 kD, as analyzed
by polyacrylamide gel electrophoresis, versus uninduced cells.
Protein lysates were used for conversion of uracil to UMP without
purification.
Reactions for CO
2
collection and distillation were performed
in flasks designed to avoid contamination by atmospheric CO
2
.
44
Reaction mixtures contained 5 mM OMP and 50 mM MOPS,
pH 7.0. Reaction mixtures were sparged with Drierite-scrubbed
N
2
prior to addition of enzyme. Reactions were initiated by the
Initial UPRTase reactions (1 mL volume) were performed using
addition of ODCase and terminated by the addition of H SO to
2
4
◦
1
2
mg unenriched uracil, 25 mM Tris-HCl, pH 8.6, 5 mM MgCl
mg BSA, as previously described. Each reaction was started
2
,
pH < 2. Partial reactions (30 mmol OMP) were carried out at 25 C
in a water bath, with enough enzyme added to convert about 30%
of the substrate to product within a few minutes. For every partial
reaction, a complete reaction (typically 5-fold amount of ODCase,
5-fold reaction time versus partial reactions) containing 10 mmol
OMP was performed, and checked for completion by HPLC. Col-
45
by addition of 5.5 mg PRPP and protein (typically 5 mL of
◦
lysate) carried out at 37 C with gentle shaking. Reactions for
1
8
[
O2]-UMP synthesis were scaled up proportionally to a total
volume of 6 mL. The appearance of UMP was monitored by
1
8
HPLC as described above. Once the conversion to [ O2]-UMP
was near completion (>95% converted), the samples were frozen
until purification could be carried out. Purification was performed
using anion exchange chromatography as described above. After
lection of CO
cryodistillation system under vacuum, similar to previous work.
The composition of the remote label OMP mixture (double-
enriched plus depleted) was adjusted so that CO generated from
2
was carried out using a specially designed glass
18
2
1
8
purification, the [ O2]-UMP was analyzed by LC-MS, as above,
a complete reaction (10 mmoles) had a delta value approximately
equal to that of the natural abundance OMP. OMP mixtures that
1
8
to ensure retention of O enrichment.
Synthesis of [ O2, C-carboxyl] OMP from [ O2]-UMP (Fig. 4,
1
8
13
18
yielded CO with a highly positive delta value, for example, were
2
Steps 4–6) was carried out as described below for natural
supplemented with more depleted OMP.
Fractions of reactions were determined following CO collection
by HPLC analysis (above) of OMP and UMP, using a UV
detection wavelength of 264 nm. At this wavelength, OMP and
UMP have equal extinction coefficients, since this is the isosbestic
1
3
13
13
abundance OMP, except that Na CN or K CN (99% C,
Cambridge Isotope Laboratories) was used in place of unenriched
NaCN. No difference could be observed in the rates or products
2
1
3
13
18
of reaction with Na CN versus K CN. After synthesis, the [ O2,
This journal is © The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 4533–4541 | 4539

View Article Online
point in the spectrophotometric assay. Peaks emerging from the
HPLC were integrated by the Breeze Software system to determine
the fraction of reaction. Using the fraction of reaction, and the
delta values for partial and complete reactions, isotope effects
were calculated for the ODCase reaction with natural abundance
OMP, and the OMP remote label mixture, using the calculations
into uracil and other compounds is gratefully acknowledged. We
thank Art Kasson, Cornell University, for analysis of standard
CO . Special thanks to Melisa Kundracik (current address: De-
2
partment of Biochemistry, Brandeis University), who composed a
lab manual on the use of the isotope ratio mass spectrometer and
CO
2
distillation line during a PRF-supported summer research
18
previously described.
project. Special thanks also to Dr. Howard Mettee, Department
of Chemistry, Youngstown State University, for keeping the CO
distillation line operating following occasional mishaps.
2
Computational methods
This work was supported by the American Chemical Society
Petroleum Research Fund, Grant #39730-B4, an instrumentation
grant from the Ohio Board of Regents Hayes Investment Fund
to the Ohio Mass Spectrometry Consortium, and partially by
grant GM63504–01 from the National Institutes of Health AREA
Program (WOW, VLS, BV, JAS).
JAS has been a program officer at ACS PRF since July 2006,
and had no affiliation with PRF at the time Grant #39730-B4 was
awarded (May 2003).
JKL and LMP gratefully acknowledge the support of NSF, the
Alfred P. Sloan Foundation, the Rutgers Busch Grant Program
and the National Center for Supercomputer Applications. We also
thank Professor Daniel Singleton for helpful discussions and use
of his version of the Quiver program.
Ground-state and transition structures were fully optimized using
B3LYP/6-31 + G* calculations as implemented in Gaussian98
46,47
and Gaussian03.
to provide reliable energetics for decarboxylation reactions.
B3LYP methods have been previously shown
10,21,48–51
A vibrational frequency analysis was performed on all stationary
points. To allow for solvation effects, structures 1, 2, 4, 6 and 8
52
were also optimized using the Onsager solvent model in water.
Equilibrium and kinetic isotope effects were calculated both in
the gas phase and in water (using the Onsager solvent-model
structures) at 298 K using the formulation of Bigeleisen and
5
3–56
Meyer.
A vibration scaling factor of 0.96 was used for both
57
the gas phase and solvent calculations. The Onsager method has
been shown to give reliable geometries and isotope effects in recent
studies of the enzyme N-methyltryptophan oxidase.
58
The effect of a hydrogen bond on O2 of OMP was explored
via a model based on the crystal structure of Bacillus subtilis
OMP decarboxylase with bound UMP. Gln194 and UMP were
References
4
extracted from an arbitrarily selected subunit of the crystal struc-
ture (1DBT). This structure was modified by replacing the sugar
moiety with a methyl group, and by reducing Gln194 to acetamide.
A carboxylate was added to UMP in an orientation consistent
with the optimized geometry of 1-methylorotate. Coordinates of
the heavy atoms originating from the crystal structure were not
altered. This hydrogen-bonded complex of 1-methylorotate and
acetamide was fully optimized in the gas phase at B3LYP/6-31 +
G* and equilibrium isotope effect calculations were conducted.
1 A. M. Rouhi, Chemical & Engineering News, 2000, 78, 42–46.
2
Orotidine monophosphate decarboxylase : a mechanistic dialogue,
Springer, Berlin, 2004.
A. Radzicka and R. Wolfenden, Science, 1995, 267, 90–93.
3
4 T. C. Appleby, C. Kinsland, T. P. Begley and S. E. Ealick, Proc. Natl.
Acad. Sci. USA, 2000, 97, 2005–2010.
5
6
7
B. G. Miller, A. M. Hassell, R. Wolfenden, M. V. Milburn and S. A.
Short, Proc. Natl. Acad. Sci. USA, 2000, 97, 2011–2016.
N. Wu, Y. Mo, J. Gao and E. F. Pai, Proc. Natl. Acad. Sci. USA, 2000,
97, 2017–2022.
P. Harris, J. C. Navarro Poulsen, K. F. Jensen and S. Larsen,
Biochemistry, 2000, 39, 4217–4224.
8
9
B. G. Miller and R. Wolfenden, Annu. Rev. Biochem., 2002, 71, 847–885.
P. Beak and B. Siegel, J. Am. Chem. Soc., 1976, 98, 3601–3606.
Conclusions
1
1
0 J. K. Lee and K. N. Houk, Science, 1997, 276, 942–945.
1 T.-S. Lee, L. T. Chong, J. D. Chodera and P. A. Kollman, J. Am. Chem.
Soc., 2001, 123, 12837–12848.
1
8
We have measured an O2 isotope effect for the ODCase
decarboxylation reaction of 0.991 ± 0.001, which leads to the
12 S. Hur and T. C. Bruice, Proc. Natl. Acad. Sci. USA, 2002, 97, 9668–
1
8
calculation of an intrinsic O effect of approximately 0.983. This
9673.
1
8
13 M. Lundberg, M. R. A. Blomberg and P. E. M. Siegbahn, J. Mol.
substantial O2 effect requires a mechanism that includes one
or more steps associated with binding and pre-decarboxylation
chemistry with significantly inverse contributions to this overall
effect. The direct decarboxylation mechanism proposes no bond
forming events on the orotate ring prior to the decarboxylation
Model., 2002, 8, 119–130.
1
1
1
4 C. L. Stanton, I.-F. W. Kuo, C. J. Mundy, T. Laino and K. N. Houk,
J. Phys. Chem. B, 2007, 111, 12573–12581.
5 K. Toth, T. L. Amyes, B. M. Wood, K. Chan, J. A. Gerlt and J. P.
Richard, J. Am. Chem. Soc., 2007, 129, 12946–12947.
6 T. L. Amyes, B. M. Wood, K. Chan, J. A. Gerlt and J. P. Richard, J. Am.
Chem. Soc., 2008, 130, 1574–1575.
17 A. Warshel, M. Strajbl, J. Villa and J. Florian, Biochemistry, 2000, 39,
4728–14738.
8 J. A. Smiley, P. Paneth, M. H. O’Leary, J. B. Bell and M. E. Jones,
1
8
step, and the calculated O2 IEs for such a mechanism do not
appear able to account for the experimental value. New structures
of the ODCase-OMP complex show an intricate network of
contacts between O2, the substrate phosphoryl group, an enzyme
Gln side chain, and a bound water molecule; this feature of the
active site appears critical for decarboxylation and may account
for the observed isotope effect data.
1
1
Biochemistry, 1991, 30, 6216–6223.
19 J. I. Ehrlich, C.-C. Hwang, P. F. Cook and J. S. Blanchard, J. Am. Chem.
Soc., 1999, 121, 6966–6967.
2
2
2
0 M. A. Rishavy and W. W. Cleland, Biochemistry, 2000, 39, 4569–4574.
1 L. M. Phillips and J. K. Lee, J. Am. Chem. Soc., 2001, 123, 12067–12073.
2 K. Tokuoka, Y. Kushakari, S. R. Krungkrai, H. Matsumura, Y. Kai, J.
Krungkrai, T. Horii and T. Inoue, J. Biochem., 2008, 143, 69–78.
3 J. G. Wittmann, D. Heinrich, K. Gasow, A. Frey, U. Diederichsen and
M. G. Rudolph, Structure, 2008, 16, 82–92.
Acknowledgements
2
2
Assistance from Dr. Roland Riesen, Youngstown State University,
in obtaining mass spectrometric analyses of isotopic incorporation
4 S. A. Barnett, T. L. Amyes, B. M. Wood, J. A. Gerlt and J. P. Richard,
Biochemistry, 2008, 47, 7785–7787.
4
540 | Org. Biomol. Chem., 2008, 6, 4533–4541
This journal is © The Royal Society of Chemistry 2008

View Article Online
2
2
5 M. H. O’Leary, in Methods in Enzymol., Academic Press, New York,
980, vol. 64, pp. 83-104.
6 W. W. Cleland, Enzyme mechanisms from isotope effects, CRC Press,
Boca Raton, FL, 2006.
M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X.
Li, J. E. Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J. Jaramillo,
R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi,
C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth,
P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D.
Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K.
Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S.
Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz,
I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y.
Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson,
W. Chen, M. W. Wong, C. Gonzalez, and J. A. Pople, GAUSSIAN03,
(2004) Gaussian, Inc., Wallingford CT.
1
2
2
7 V. L. Schramm, Curr. Opin. Chem. Biol., 2007, 11, 529–536.
8 J. D. Hermes, S. W. Morrical, M. H. O’Leary and W. W. Cleland,
Biochemistry, 1984, 23, 5479–5488.
2
3
9 S. Szapiro and F. Steckel, Trans. Faraday Soc., 1967, 63, 883–894.
0 E. Gawlita, W. S. Caldwell, M. H. O’Leary, P. Paneth and V. E.
Anderson, Biochemistry, 1995, 34, 2577–2583.
3
3
1 E. Gawlita, P. Paneth and V. E. Anderson, Biochemistry, 1995, 34,
6
050–6058.
2 T. Pineda, R. Callender and S. D. Schwartz, Biophys. J., 2007, 93,
47 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, V. G. Zakrzewski, J. A. J. Montgomery, R. E.
Stratmann, J. C. Burant, S. Dapprich, J. M. Millam, A. D. Daniels,
K. N. Kudin, M. C. Strain, O. Farkas, J. Tomasi, V. Barone, M.
Cossi, R. Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford,
J. Ochterski, G. A. Petersson, P. Y. Ayala, Q. Cui, K. Morokuma,
D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J.
Cioslowski, J. V. Ortiz, A. G. Baboul, B. B. Stefanov, G. Liu, A.
Liashenko, P. Piskorz, I. Komaromi, R. Gomperts, R. L. Martin,
D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, C.
Gonzalez, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W.
Wong, J. L. Andres, C. Gonzalez, M. Head-Gordon, E. S. Replogle,
and J. A. Pople,GAUSSIAN98, (1998) Gaussian, Inc., Pittsburgh, PA.
48 D. A. Singleton, S. A. Merrigan, B. J. Kim, P. Beak, L. M. Phillips and
J. K. Lee, J. Am. Chem. Soc., 2000, 122, 3296–3300.
1
474–1483.
3
3
3 A. Sievers and R. Wolfenden, Bioorg. Chem., 2005, 33, 45–52.
4 T. L. Amyes, J. P. Richard and J. J. Tait, J. Am. Chem. Soc., 2005, 127,
1
5708–15709.
3
3
3
3
5 J. A. Smiley, K. M. Hay and B. S. Levison, Bioorg. Chem., 2001, 29,
9
6–106.
6 K. N. Houk, J. K. Lee, D. J. Tantillo, S. Bahmanyar and B. N. Hietbrink,
Chem. BioChem., 2001, 2, 113–118.
7 C. L. J. Turnbough, K. H. Kerr, W. R. Funderburg, J. P. Donahue and
F. E. Powell, J. Biol. Chem., 1987, 262, 10239–10245.
8 Current protocols in molecular biology, Greene Publishing Associates
and Wiley-Interscience, New York, 1994.
3
4
4
4
9 B. Vemulapalli, M. S. Thesis, Youngstown State University, 2006.
0 R. S. Brody and F. H. Westheimer, J. Biol. Chem., 1979, 254, 4238–4244.
1 J. B. Bell and M. E. Jones, J. Biol. Chem., 1991, 266, 12662–12667.
2 T. Ueda, M. Yamamoto, A. Yamane, M. Imazawa and H. Inoue,
J. Carbohydr. Nucleosides, Nucleotides, 1978, 5, 261–271.
49 L. M. Phillips and J. K. Lee, J. Org. Chem., 2005, 70, 1211–1221.
50 R. D. Bach and C. Canepa, J. Am. Chem. Soc., 1997, 119, 11725–11733.
51 R. D. Bach, C. Canepa and M. N. Glukhovtsev, J. Am. Chem. Soc.,
1999, 121, 6542–6555.
4
3 P. S. Anderson, J. M. Smith and B. Mygind, Eur. J. Biochem., 1992, 204,
5
1–56.
52 L. Onsager, J. Am. Chem. Soc., 1936, 58, 1486–1493.
4
4
4 V. L. Smiley, M. S. Thesis, Youngstown State University, 2006.
5 H. K. Jensen, N. Mikkelsen and J. Neuhard, Protein Expression and
Purification, 1997, 10, 356–364.
53 J. Bigeleisen and M. G. Mayer, J. Chem. Phys., 1947, 15, 261–267.
54 M. Wolfsberg, Acc. Chem. Res., 1972, 5, 225–233.
55 M. Saunders, K. E. Laidig and M. Wolfsberg, J. Am. Chem. Soc., 1989,
111, 8989–8994.
56 D. A. Singleton, Personal Communication.
57 A. P. Scott and L. Radom, J. Phys. Chem. B, 1996, 100, 16502–16513 .
58 E. C. Ralph, J. S. Hirschi, M. A. Anderson, W. W. Cleland, D. A.
Singleton and P. F. Fitzpatrick, Biochemistry, 2007, 46, 7655–7664.
4
6 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, J. A. J. Montgomery, T. Vreven, K. N. Kudin,
J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B.
Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H.
Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa,
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