Enzyme Architecture: Erection of Active Orotidine 5′-Monophosphate Decarboxylase by Substrate-Induced Conformational Changes

Article abstract of DOI:10.1021/jacs.7b08897

Orotidine 5′-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with k_cat/K_m = 1.4 × 10^-7 M^-1 s^-1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change.

Full text of DOI:10.1021/jacs.7b08897

Subscriber access provided by the Henry Madden Library | California State University, Fresno
Communication
Enzyme Architecture: Erection of Active Orotidine 5'-Monophos-
phate Decarboxylase by Substrate-Induced Conformational Changes
Archie C. Reyes, Tina L. Amyes, and John Richard
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08897 • Publication Date (Web): 23 Oct 2017
Downloaded from http://pubs.acs.org on October 27, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
Enzyme Architecture: Erection of Active Orotidine 5'-Monophos-
phate Decarboxylase by Substrate-Induced Conformational Changes
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*
Archie C. Reyes, Tina L. Amyes and John P. Richard
Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York, 14260ꢀ3000, United States
Supporting Information Placeholder
tion state for decarboxylation of the truncated substrate
ABSTRACT: Orotidine 5'ꢀmonophosphate decarboxꢀ
piece 1ꢀ(βꢀDꢀerythrofuranosyl)orotate (EO) from inꢀ
ylase (OMPDC) catalyzes the decarboxylation of 5ꢀ
ꢀ7
ꢀ1 ꢀ1
6ꢀ7
fluoroorotate (FO) with k /K = 1.4 x 10
M
s .
teractions with 1.0 M phosphite dianion (Figure 1A).
cat
m
This activation is due to the utilization of binding enꢀ
ergy from interactions between OMPDC and activator
(Act, Figure 1) to drive a complex conformational
Combining this and related kinetic parameters shows
that the 31 kcal/mol stabilization of the transition state
for decarboxylation of OMP provided by OMPDC repꢀ
resents the sum of 11.8 and 10.6 kcal/mol stabilization
by the substrate phosphodianion and the ribosyl ring,
respectively, and an 8.6 kcal/mol stabilization from the
orotate ring. The transition state for OMPDCꢀcatalyzed
decarboxylation of FO is stabilized by 5.2, 7.2 and 9.0
kcal/mol, respectively, by 1.0 M phosphite dianion, Dꢀ
glycerol 3ꢀphosphate and Dꢀerythritol 4ꢀphosphate.
The stabilization is due to the utilization of binding
interactions of the substrate fragments to drive an enꢀ
zyme conformational change, which locks the orotate
ring of the whole substrate, or the substrate pieces in a
caged complex. We propose that enzymeꢀactivation is
a possible, and perhaps probable, consequence of any
substrateꢀinduced enzyme conformational change.
change from inactive open OMPDC (E ) to the active
O
8
closed caged complex (E ), where E is stabilized
C C
relative to E by interactions between dianions and the
O
9
side chains of Q215 Y217 and R235.
Scheme 1
The underlying cause for enzymatic catalysis is staꢀ
bilization of the transition state by interactions with the
1
protein catalyst. Orotidine 5'ꢀmonophosphate decarꢀ
boxylase (OMPDC) effects a 31 kcal/mol stabilization
of the transition state for decarboxylation of orotidine
5
5
'ꢀmonophosphate
'ꢀmonophosphate (UMP), by a stepwise mechanism
(OMP)
to
give
uridine
2
3
through a UMP carbanion intermediate (Scheme 1).
This transition state stabilization has been modeled in
computational studies. What has not been fully modꢀ
4
Figure 1. Activation of OMPDCꢀcatalyzed decarboxylaꢀ
tion reactions. (A) Activation of decarboxylation of EO
eled is the extraordinary specificity of OMPDC in
binding the decarboxylation transition state with a
higher affinity (31 kcal/mol) compared with the subꢀ
2
ꢀ 6, 10
and FEO by HPO .
(B) Activation of decarboxylaꢀ
3
2ꢀ
tion of FO by HPO . (C, D) Activation of decarboxylaꢀ
3
2, 5
tion of FO by Dꢀglycerol 3ꢀphosphate (DG3P) and Dꢀ
erythritol 4ꢀphosphate (DE4P), respectively.
strate OMP (8 kcal/mole).
Binding interactions between OMPDC and the phosꢀ
phodianion of OMP provide 12 of the 31 kcal/mol
Figure 2A shows a representation of the open form
of OMPDC determined for unliganded OMPDC from
6
transition state stabilization. These interactions do not
only anchor OMP to the protein, since eliminating the
anchoring connection results in only a 4 kcal/mol deꢀ
crease, to 8 kcal/mol, in the stabilization of the transiꢀ
yeast (E , Scheme 2), with a hypothetical 6ꢀaza uriꢀ
O
dine 5'ꢀmonophosphate (azaUMP) ligand inserted at
the position determined for the OMPDC•azaUMP
11
complex (Figure 2B). Many ligands induce a large
ACS Paragon Plus Environment

Journal of the American Chemical Society
conformational change in OMPDC that is driven by the was determined as the slope of the linear plot of kobs
Page 2 of 5
=
1
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
development of strong proteinꢀligand interactions.
The interactions between the protein and the phosꢀ
phodianion, or the ribosyl hydroxyls, which develop at
the complex to the tightꢀbinding inhibitor azaUMP [or
v[E] against [FO] (Figure 3A). These apparent firstꢀ
order rate constants from HPLC analyses are reproducꢀ
ible to better than ±10%. The 5ꢀF substituent provides
strong stabilization of the UMP carbanion intermediate
12
3a, 10, 13b, 14
to substrate analogs], are illustrated in Figure 2B for
of OMPDCꢀcatalyzed reactions.
The value of
11
ꢀ10
ꢀ1 ꢀ1
the closed form of OMPDC (EC). Our model
(k /K ) = 3 x 10
M
s
for OMPDCꢀcatalyzed
cat m o
(
Scheme 2) requires that the binding energy from all
decarboxylation of orotate (Table 1) was determined
ꢀ7
ꢀ1 ꢀ1
interactions that drive the conformational change from
from (k /K ) = 1.4 x 10 M s for decarboxylation
cat m o
E to E activate OMPDC for catalysis, as has been
demonstrated for proteinꢀdianion interactions,
because these interactions are only fully expressed at
the decarboxylation transition state. This prediction is
confirmed here by the observation that binding interacꢀ
tions between OMPDC and either phosphite dianion
of FO and a 500ꢀfold effect of the 5ꢀF substituent.
This is a rough average of the 5ꢀF effect on mutant
OMPDCꢀcatalyzed decarboxylation of OMP, when
chemistry is strongly rate determining for both
O
C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6, 10, 13
1
4
OMPDCꢀcatalyzed reactions.
Scheme 2
(
Figure 1B) or sugar hydroxyls (Figure 1C and 1D)
activate OMPDC for catalysis of decarboxylation of
the definitive truncated substrate, 5ꢀfluoroorotate (FO).
A
B
Figure 2. Representations of the open EO (PDB entry
3GDK) and the closed, liganded, (B, 3GDL) forms of
11
yeast OMPDC. The azaUMP ligand is placed at strucꢀ
ture A at the position determined for structure B. The ligꢀ
and is stabilized by interactions from the side chains of
R235 and Q215 with the phosphodianion, and of the D96,
H61, D37 and K59 with the ribosyl hydroxyls.
The slow decarboxylation of FO to form
5
ꢀfluorouracil (FU) catalyzed by yeast OMPDC was
monitored by HPLC analyses, as described in the Supꢀ
porting Information (SI) to this manuscript. Observed
firstꢀorder rate constants kobs = v/[E] for the decarboxyꢀ
lation of FO (5 or 10 mM) catalyzed by 0.7 mM
OMPDC at 25 °C were determined from the initial
reaction velocity v during the first 0.01% reaction, obꢀ
tained over a twoꢀweek reaction time, during which
OMPDC maintained full activity. The secondꢀorder
Figure 3. Plots of kinetic data for OMPDCꢀcatalyzed
decarboxylation of FO. (A) The dependence of kobs = v[E]
on [FO]. (B) The dependence of (k /K ) for OMPDCꢀ
cat m obs
2ꢀ
catalyzed decarboxylation of FO on [HPO ]. (C) The
3
dependence of (k /K ) for OMPDCꢀcatalyzed decarꢀ
cat m obs
boxylation of FO on the concentration of DE4P
A comparison of the secondꢀorder rate constants
for OMPDCꢀcatalyzed decarboxylation of oroꢀ
.
ꢀ7
ꢀ1 ꢀ1
rate constant (k /K ) = (1.4 ± 0.1) x 10 M s for
cat m o
OMPDCꢀcatalyzed decarboxylation of FO (Table 1)
kcat/K
m
ACS Paragon Plus Environment

Page 3 of 5
Journal of the American Chemical Society
driven protein conformational change on the reactivity
tate, EO, and OMP (Table 1) show that the ribosyl and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
phosphodianion fragments contribute 10.6 and 11.8
kcal/mol intrinsic binding energies, respectively, to
stabilization of the transition state for OMPDCꢀ
catalyzed decarboxylation of OMP: the sum is 22.4
kcal/mol of the total 31 kcal/mol total intrinsic binding
energy of OMP, leaving 8.6 kcal/mole for stabilization
of the fluoroorotate ring is transmitted across both the
ribosyl group and the vacant protein core. DG3P and
DE4P provide 7.2 and 9.0 kcal/mol transition state staꢀ
bilization, respectively, which corresponds to ca 2
kcal/mol transition state stabilization/sugar hydroxyl.
OMPDC shows specificity for activation by the DG3P,
since the IBE determined for LG3P is similar to that
2
of the transition state by interactions with orotate.
2ꢀ
This provides a graphic and readily generalizable ilꢀ
lustration of how enormous enzymatic rate acceleraꢀ
tions may be obtained through recruitment of modest,
additive, binding energies of substrate fragments.
for HPO3 alone. The small activation of OMPDCꢀ
catalyzed decarboxylation of FO for a reaction in the
presence of 40 mM Dꢀribose 5'ꢀphosphate (DR5P, SI)
is consistent with a ligand IBE of only 3.1 kcal/mol
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Table 2). This shows that a tight and precise fit of the
Table 1. Contribution of the IBE from Substrate Frag-
10
2
a
ments to the 31 kcal/mol IBE of OMP for OMPDC
ligand is required for strong transition state binding.
Table 2. Kinetic Parameters for Unactivated and Acti-
a
k /K
M s )
c
Fragment IBE
(kcal/mole)
vated OMPDC-Catalyzed Decarboxylation of FO
cat
m
Substrate
ꢀ1 ꢀ1 b
Fragment
H
d
(
(1.4 ± 0.1) x
(k /K )
[(k /K ) ]/Kd
IBE
(kcal/mol)
cat
m
Act
c
cat
m
Act
FO
Orotate
EO
ꢀ7 e
Activator
d
K (M)
ꢀ1
ꢀ1 b
ꢀ2 ꢀ1 d
e
1
0
M
s
M
s
ꢀ10 f
3 x 10
0
f
(1.6 ± 0.4)
x 10
0.18 ±
0.05
0.05
ꢀ4
HP
i
ꢀ4
(8.4 ± 0.4) x 10
5.2
6.8
g
(7.0 ± 0.7)
ꢀ2
ꢀ4
0.02
DꢀErythrose
10.6
D,LG3P
ꢀ4
(1.3 ± 0.1) x 10
(9.9 ± 0.2) x 10
x 10
±0.01
g, h
LG3P
5.2
7.2
7
Ribose 5'ꢀ
Phosphate
i
ꢀ2
OMP
1 x 10
10.6 + 11.8
DG3P
DE4P
DR5P
(2.5 ± 0.01) x 10
j
(1.9 ± 0.2)
0.030 ±
0.003
ꢀ1
a
b
(6.0 ± 0.2) x 10
9.0
3.1
ꢀ2
At 25 °C, pH 7.0 and I = 0.14–0.15 (NaCl). Secondꢀ
x 10
g
ꢀ5
order rate constant for decarboxylation catalyzed by
≈ 3 x 10
c
d
a
OMPDC. Fragment attached to orotate. Contribution
of fragment to the stabilization of the transition state for
At 25 °C, pH 7.0 and I = 0.15 (NaCl). The quoted errors
are the standard deviations from the leastꢀsquares fits of
e
b
OMPDC catalyzed decarboxylation of OMP. Figure 3A.
the data. Secondꢀorder rate constant for breakdown of
f
c
See text.
E•Act•FO to form FU. Dissociation constant for the
d
e
activator. Determined as described in the text. Fragꢀ
Figures 3B and 3C show the effect of increasing
‡
2
ꢀ
ment intrinsic binding energy, calculated as ꢀRTlnK (eq
). Figure 3B. See SI. No detectable saturation of
[
HPO ] and [DE4P], respectively, on (k /K ) for
3 cat m obs
f
g
h
3
OMPDCꢀcatalyzed decarboxylation of FO (5 mM).
These data were fit to eq 1 (Scheme 3A) to give the
values for (k /K ) and K reported in Table 2. Table
i
OMPDC.
Calculated from eq 2, where (kAct) =
j
[
(k /K ) ]/K from Scheme 3A. Figure 3C.
cat m Act
d
cat m Act
d
2
reports values for (k /K ) /K obtained as the slope
Scheme 3
cat m Act
d
of the linear correlations shown as dashed lines in Figꢀ
ures 3B and 3C. Data for the effect of increasing
[D,LG3P] (Figure S1A) and [LG3P] (Figure S1B) on
(kcat/Km)obs for OMPDCꢀcatalyzed decarboxylation of
FO were evaluated to give the kinetic parameters in
Table 2. The value of (k /K ) /K for OMPDCꢀ
cat m Act
d
catalyzed decarboxylation of DG3P was calculated
from these kinetic parameters using eq 2, where
(
k )
= [(k /K ) /K )]
(X = D, L, or D, L)
Act XG3P
cat m Act
d
XG3P
The intrinsic fragment binding energies (IBEs), deterꢀ
mined using eq 3 (Scheme 3B), are reported in Table 2.
1
0
Phosphite dianion (1.0 M) provides similar 5.0 and
5.2 (Table 2) kcal/mol stabilization, respectively, of the
transition states for OMPDCꢀcatalyzed decarboxylaꢀ
tion of truncated substrates 1ꢀ(βꢀDꢀerythrofuranosyl)ꢀ
ꢄ
ꢋ
(
ꢀꢁꢂꢃ/ꢄ ) ꢉ=ꢉꢊ
ꢉꢐ (ꢀꢁꢂꢃ/ꢄ ) +
ꢅ
ꢄ ꢉꢌꢉꢍꢎꢁꢃꢏ
ꢆ
ꢅ
ꢆꢇꢈ
ꢋ
ꢍꢎꢁꢃꢏ
ꢉꢐ (ꢀꢁꢂꢃ/ꢄ )ꢎꢁꢃꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉ
ꢑꢒꢓ
ꢅ
ꢄ ꢉꢌꢉꢍꢎꢁꢃꢏ
5
ꢀfluoroorotate (FEO) and FO. The small apparent
ꢉꢊ
ꢋ
effect of the ribosyl group of FEO on activation for
decarboxylation shows that the effect of the dianion
ꢑꢀꢎꢁꢃꢓꢔꢕꢖꢗ =ꢉꢘꢑꢀꢎꢁꢃꢓꢔ,ꢙꢕꢖꢗ ꢉꢉꢉ−ꢉꢑꢀꢎꢁꢃꢓꢙꢕꢖꢗꢉꢉꢉꢉꢉꢉꢑꢘꢓ
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 5
ꢚꢠ (ꢡ /ꢚ )
ꢞ
ꢜꢢꢝ
ꢣ ꢤꢥꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢉꢑ3ꢓ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢚ
ꢛꢜꢝ
ꢉ=ꢉꢟ
AUTHOR INFORMATION
(
ꢡꢜꢢꢝ/ꢚꢣ)ꢛꢜꢝ
Corresponding Author
EMAIL: jrichard@buffalo.edu. Tel: (716)645ꢀ4232
These results provide strong support for the concluꢀ
sion that each of the many OMPDCꢀsubstrate interacꢀ
tions, which stabilize the closed enzyme E relative to
*
Notes
C
The authors declare no competing financial interests.
the open enzyme E , contribute to activation of
O
OMPDC for catalysis of decarboxylation of orotate
and fluoroorotate rings. We conclude that these protein
ligand interactions act in concert to construct a tight,
catalytically active form of OMPDC from the floppy
open enzyme. These results provide a dramatic examꢀ
ple of the effect of such preorganization of protein
ACKNOWLEDGMENT
This work was supported by grants GM116921 and
GM39754 from the National Institutes of Health.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
REFERENCES
1. (a) Pauling, L., Nature 1948, 161, 707-709; (b) Amyes, T.
L.; Richard, J. P., Biochemistry 2013, 52, 2021-2035.
15
structure on enzyme activity.
2. Radzicka, A.; Wolfenden, R., Science 1995, 267, 90-93.
Xꢀray crystallographic analyses and other protocols
for the evaluation of the role of conformational changꢀ
es in enzyme catalysis, failed to suggest the activating
nature of phosphodianion driven conformational
changes in catalysis by triosephosphate isomerase, and
3. (a) Tsang, W.-Y.; Wood, B. M.; Wong, F. M.; Wu, W.; Gerlt,
J. A.; Amyes, T. L.; Richard, J. P., J. Am. Chem. Soc. 2012, 134,
4580-14594; (b) Amyes, T. L.; Wood, B. M.; Chan, K.; Gerlt, J.
A.; Richard, J. P., J. Am. Chem. Soc. 2008, 130, 1574-1575.
. (a) Vardi-Kilshtain, A.; Doron, D.; Major, D. T., Biochemistry
1
4
2013, 52, 4382-4390; (b) Wu, N.; Mo, Y.; Gao, J.; Pai, E. F.,
Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 2017-2022; (c) Warshel,
A.; Strajbl, M.; Villa, J.; Florian, J., Biochemistry 2000, 39, 14728-
7b, 16
other enzymes.
Our report of the, likewise, unrecꢀ
ognized activating role of protein sugarꢀhydroxyl interꢀ
actions in catalysis by OMPDC provides compelling
motivation for a reevaluation of the role of substrateꢀ
1
1
2
4738; (d) Gao, J., Curr. Op. Struct. Biol. 2003, 13, 184-192.
5. Porter, D. J. T.; Short, S. A., Biochemistry 2000, 39, 11788-
1800.
6. Amyes, T. L.; Richard, J. P.; Tait, J. J., J. Am. Chem. Soc.
005, 127, 15708-15709.
1b,
driven conformational changes in enzyme catalysis;
8a, 8b, 17
and, for experiments to test the hypothesis that
7. (a) Spong, K.; Amyes, T. L.; Richard, J. P., J. Am. Chem.
Soc. 2013, 135, 18343-18346; (b) Reyes, A. C.; Zhai, X.;
Morgan, K. T.; Reinhardt, C. J.; Amyes, T. L.; Richard, J. P., J.
Am. Chem. Soc. 2015, 137, 1372-1382.
8. (a) Amyes, T. L.; Malabanan, M. M.; Zhai, X.; Reyes, A. C.;
Richard, J. P., Prot. Eng. Des. & Sel. 2017, 30, 157-165; (b)
Richard, J. P.; Amyes, T. L.; Goryanova, B.; Zhai, X., Curr. Op.
Chem. Biol. 2014, 21, 1-10; (c) Go, M. K.; Amyes, T. L.; Richard,
J. P., Biochemistry 2009, 48, 5769-5778.
many, or most, such protein conformational changes
activate enzymes for catalysis of the reaction of their
bound substrates.
Experiments to probe the activation of OMPDC by
substrate induced conformational changes have outꢀ
paced other mechanistic studies on this enzyme. Conꢀ
sequently, there is now strong evidence that the bindꢀ
ing interactions of the nonreacting substrate parts are
used to construct a protein cage that shows a high reacꢀ
tivity towards substrate decarboxylation. The results of
our earlier work have emphasized the role the protein
plays in strongly stabilizing the UMP carbanion interꢀ
9
. (a) Amyes, T. L.; Ming, S. A.; Goldman, L. M.; Wood, B. M.;
Desai, B. J.; Gerlt, J. A.; Richard, J. P., Biochemistry 2012, 51,
630-4632; (b) Goldman, L. M.; Amyes, T. L.; Goryanova, B.;
Gerlt, J. A.; Richard, J. P., J. Am. Chem. Soc. 2014, 136, 10156-
0165.
0. Goryanova, B.; Spong, K.; Amyes, T. L.; Richard, J. P.,
4
1
1
Biochemistry 2013, 52, 537-546.
11. Chan, K. K.; Wood, B. M.; Fedorov, A. A.; Fedorov, E. V.;
Imker, H. J.; Amyes, T. L.; Richard, J. P.; Almo, S. C.; Gerlt, J. A.,
Biochemistry 2009, 48, 5518-5531.
3
mediate relative to the carbon acid substrate. This
suggests that an unusually strong stabilization of this
carbanion by interaction with the cationic side chain of
12. (a) Fujihashi, M.; Wei, L.; Kotra, L. P.; Pai, E. F., J. Mol.
Biol. 2009, 387, 1199-1210; (b) Wittmann, J. G.; Heinrich, D.;
Gasow, K.; Frey, A.; Diederichsen, U.; Rudolph, M. G., Structure
4
c, 16a
K93.
This single interaction seems unlikely to
2008, 16, 82-92.
enable the entire rate acceleration, so that there reꢀ
mains much to be learned about the origin of the highꢀ
reactivity of the caged OMPDCꢀsubstrate complex
13. (a) Goryanova, B.; Goldman, L. M.; Amyes, T. L.; Gerlt, J.
A.; Richard, J. P., Biochemistry 2013, 52, 7500-7511; (b)
Goryanova, B.; Amyes, T. L.; Gerlt, J. A.; Richard, J. P., J. Am.
Chem. Soc. 2011, 133, 6545-6548.
14. Goryanova, B.; Goldman, L. M.; Ming, S.; Amyes, T. L.;
Gerlt, J. A.; Richard, J. P., Biochemistry 2015, 54, 4555-4564.
ASSOCIATED CONTENT
15. Warshel, A., J. Biol. Chem 1998, 273, 27035-27038.
16. (a) Miller, B. G.; Hassell, A. M.; Wolfenden, R.; Milburn, M.
Supporting Information. References for experimental proceꢀ
dures used to purify yeast OMPDC, and a description of the
procedures for the assay of OMPDCꢀcatalyzed decarboxylation
reaction of FO. Kinetic data for the activation of OMPDC by Dꢀ
ribose 5'ꢀphosphate. Figures S1A and S1B that show the effect
on increasing [LG3P] and [D,LG3P], respectively on (k /K )
for OMPDCꢀcatalyzed decarboxylation of FO. This material is
available free of charge via the Internet at http://pubs.acs.org.
V.; Short, S. A., Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 2011-
2016; (b) Ou, X.; Ji, C.; Han, X.; Zhao, X.; Li, X.; Mao, Y.; Wong,
L.-L.; Bartlam, M.; Rao, Z., J. Mol. Biol. 2006, 357, 858-869; (c)
Davenport, R. C.; Bash, P. A.; Seaton, B. A.; Karplus, M.; Petsko,
G. A.; Ringe, D., Biochemistry 1991, 30, 5821-6; (d) Kholodar, S.
A.; Allen, C. L.; Gulick, A. M.; Murkin, A. S., J. Am. Chem. Soc.
2015, 137, 2748-2756.
cat m obs
17. Malabanan, M. M.; Amyes, T. L.; Richard, J. P., Curr. Op.
Struct. Biol. 2010, 20, 702-710.
ACS Paragon Plus Environment

Page 5 of 5
Journal of the American Chemical Society
INSERT TABLE OF CONTENTS ARTWORK HERE [3.5 X 9.0 CM]
1
2
3
4
5
6
7
8
9
Orotidine 5’-Monophosphate Decarboxylase
Intrinsic Binding Energy
Whole Substrate
O
H+ CO
Act +
O
Act +
2
pyrimidine
X
X
HN
HN
O
8.6 kcal/mol
dianion
1.8 kcal/mol
O
N
R
CO
OMPDC
2
O
N
R
H
HN
1
2
O
N
CO
2
2
O
2
POH C
3
OH
OH OH
O
O
O
3
POH
2
C
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
P
H
O
2
P H
2-O
3
PO
O
Act
O
O
O
OH
H
OH OH
O
R
R
=
H
R
=
H
R
=
H
ribosyl
ring
10.6 kcal/mol
OH OH
X
=
=
H
F
F
F
F
IBE (Act)
7.7 5.0
5.2
7.2
9.0 kcal/mol
ACS Paragon Plus Environment