Mechanism of the orotidine 5′-monophosphate decarboxylase-catalyzed reaction: Importance of residues in the orotate binding site

Article abstract of DOI:10.1021/bi2012355

The reaction catalyzed by orotidine 5′-monophosphate decarboxylase (OMPDC) is accompanied by exceptional values for rate enhancement (k _cat/k_non = 7.1 ± 10¹⁶) and catalytic proficiency [(k_cat/K_M)/k_non = 4.8 ± 10²² M^-1]. Although a stabilized vinyl carbanion/carbene intermediate is located on the reaction coordinate, the structural strategies by which the reduction in the activation energy barrier is realized remain incompletely understood. This laboratory recently reported that "substrate destabilization" by Asp 70 in the OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the activation energy barrier by ～5 kcal/mol (contributing ～2.7 ± 10³ to the rate enhancement) [Chan, K. K., Wood, B. M., Fedorov, A. A., Fedorov, E. V., Imker, H. J., Amyes, T. L., Richard, J. P., Almo, S. C., and Gerlt, J. A. (2009) Biochemistry 48, 5518-5531]. We now report that substitutions of hydrophobic residues in a pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutral hydrophilic residues decrease the value of k_cat by as much as 400-fold but have a minimal effect on the value of k_ex for exchange of H6 of the FUMP product analogue with solvent deuterium; we hypothesize that this pocket destabilizes the substrate by preventing hydration of the substrate carboxylate group. We also report that substitutions of Ser 127 that is proximal to O4 of the orotate ring decrease the value of k _cat/K_M, with the S127P substitution that eliminates hydrogen bonding interactions with O4 producing a 2.5 ± 10 ⁶-fold reduction; this effect is consistent with delocalization of the negative charge of the carbanionic intermediate on O4 that produces an anionic carbene intermediate and thereby provides a structural strategy for stabilization of the intermediate. These observations provide additional information about the identities of the active site residues that contribute to the rate enhancement and, therefore, insights into the structural strategies for catalysis.

Full text of DOI:10.1021/bi2012355

Article
pubs.acs.org/biochemistry
Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-
Catalyzed Reaction: Importance of Residues in the Orotate Binding
Site
†
†
‡
‡
‡
Vanessa Iiams, Bijoy J. Desai, Alexander A. Fedorov, Elena V. Fedorov, Steven C. Almo,
,†
and John A. Gerlt*
†
Departments of Biochemistry and Chemistry, University of Illinois, Urbana, Illinois 61801, United States
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States
‡
ABSTRACT: The reaction catalyzed by orotidine 5′-monophosphate decarbox-
ylase (OMPDC) is accompanied by exceptional values for rate enhancement (k /
cat
16
22
−1
knon = 7.1 × 10 ) and catalytic proficiency [(k /K )/k = 4.8 × 10 M ].
cat
M
non
Although a stabilized vinyl carbanion/carbene intermediate is located on the
reaction coordinate, the structural strategies by which the reduction in the
activation energy barrier is realized remain incompletely understood. This
laboratory recently reported that “substrate destabilization” by Asp 70 in the
OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the
3
activation energy barrier by ∼5 kcal/mol (contributing ∼2.7 × 10 to the rate
enhancement) [Chan, K. K., Wood, B. M., Fedorov, A. A., Fedorov, E. V., Imker,
H. J., Amyes, T. L., Richard, J. P., Almo, S. C., and Gerlt, J. A. (2009) Biochemistry
4
8, 5518−5531]. We now report that substitutions of hydrophobic residues in a
pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutral hydrophilic residues
decrease the value of kcat by as much as 400-fold but have a minimal effect on the value of k for exchange of H6 of the FUMP
ex
product analogue with solvent deuterium; we hypothesize that this pocket destabilizes the substrate by preventing hydration of
the substrate carboxylate group. We also report that substitutions of Ser 127 that is proximal to O4 of the orotate ring decrease
6
the value of k /K , with the S127P substitution that eliminates hydrogen bonding interactions with O4 producing a 2.5 × 10 -
cat
M
fold reduction; this effect is consistent with delocalization of the negative charge of the carbanionic intermediate on O4 that
produces an anionic carbene intermediate and thereby provides a structural strategy for stabilization of the intermediate. These
observations provide additional information about the identities of the active site residues that contribute to the rate
enhancement and, therefore, insights into the structural strategies for catalysis.
3,4
he reaction catalyzed by orotidine 5′-monophosphate
gen, although at rates that are slower than those observed for
decarboxylation of either OMP or 5-fluoroOMP. However, the
structural strategies by which the activation energy barrier
(ΔG ) for decarboxylation of OMP is reduced by 23 kcal/mol
have not yet been completely described.
T
decarboxylase (OMPDC) continues to attract attention
⧧
Scheme 1
Although “ground state destabilization” of the substrate’s
carboxylate group by its presumed proximity to Asp 70 in a
conserved hydrogen-bonding network (residue numbering for
MtOMPDC) was proposed when structures for four OMPDCs
5
6
were reported in 2000 (MtOMPDC, ScOMPDC, and the
7
8
OMPDCs from Bacillus subtilis and Escherichia coli ), that
because the structural bases for its impressive rate enhancement
9
−11
1
6
proposal has been criticized.
Recently, we provided
(
=
k /k = 7.1 × 10 ) and catalytic proficiency [(k /K )/k
cat non
cat
M
non
22
−1
1
experimental evidence of “substrate destabilization” in which
4.8 × 10 M ] are not well understood. A “vinyl carbanion”
destabilizing interactions develop as the reaction coordinate is
intermediate is on the reaction coordinate (Scheme 1). The
reactions catalyzed by the OMPDCs from Saccharomyces
cerevisiae (ScOMPDC) and Methanothermobacter thermoauto-
trophicus (MtOMPDC) proceed with a product isotope effect
of 1.0 (measured when the reaction is conducted in a 1:1
traversed (the value of the K for the OMP is less than the K
M
i
for the UMP product in which the carboxylate group is absent,
4
thereby discounting ground state destabilization). Substitution
of Asp 70 with an isosteric uncharged residue (D70N) or a less
2
mixture of H O and D O). Both enzymes also catalyze the
2
2
exchange of H6 of the UMP product and the activated 5-
fluoroUMP (FUMP) product analogue with solvent hydro-
Received: August 8, 2011
Published: August 26, 2011
©
2011 American Chemical Society
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Article
Table 1. Data Collection and Refinement Statistics for azaUMP Complexes of MtOMPDC Mutants
S127G
S127A
S127P
Data Collection
P21
I96S
I96T
L123N
L123S
P21
V155S
space group
P21
P21
P41
P21
P21
P21
no. of molecules in the
asymmetric unit
2
2
2
2
2
2
2
2
cell dimensions
a (Å)
59.78
63.64
61.12
115.20
1.45
59.71
63.60
61.20
115.19
1.32
56.67
56.67
125.95
59.79
64.09
61.60
115.60
1.30
59.78
63.95
61.64
115.47
1.3
59.78
64.14
61.61
115.55
1.3
59.77
64.04
61.61
115.50
1.3
59.71
63.83
61.70
115.22
1.3
b (Å)
c (Å)
β (deg)
resolution (Å)
no. of unique reflections
Rmerge
1.30
68702
0.064
93.4
96918
0.059
99.8
91113
0.071
93.6
99507
0.092
96.3
97354
0.083
94.6
101238
0.083
98.2
97097
0.074
94.3
92236
0.069
89.4
completeness (%)
Refinement
resolution (Å)
Rcryst
25.0−1.45
0.184
25.0−1.32
0.196
25.0−1.30
0.234
25.0−1.30
0.223
25.0−1.3
0.161
25.0−1.30
0.161
25−1.3
0.160
25−1.3
0.175
Rfree
0.216
0.218
0.258
0.247
0.183
0.175
0.169
0.193
no. of atoms
protein
3296
522
3396
405
3294
308
3378
348
3404
463
3464
482
3410
419
3402
398
water
bound ligand
no. of ligand atoms
rmsd
6azaUMP
42
6azaUMP
42
6azaUMP
42
6azaUMP
42
6azaUMP
42
6azaUMP
42
6azaUMP
42
6azaUMP
42
bond lengths (Å)
bond angles (deg)
PDB entry
0.004
1.2
0.006
1.0
0.005
1.2
0.006
1.1
0.006
1.1
0.006
1.1
0.006
1.1
0.006
1.1
3LLD
3SY5
3LLF
3PBU
3RPV
3PBW
3PBY
3PC0
sterically demanding, uncharged residue (D70G) reduces the
liganded complexes with anionic intermediate analogues (BMP
and azaUMP), O4 of the OMP substrate is assumed to
participate in hydrogen bonding interactions with the backbone
amide of Ser 127 as well as a structurally conserved water
molecule. Delocalization of the negative charge on O4 as the
anionic transition state/intermediate is formed should increase
value of kcat for decarboxylation by a factor of ∼2700 (D70G)
without significantly affecting the value of k for exchange of
ex
H6 of FUMP with solvent hydrogen.
Wolfenden estimated the value of the pK of H6 of the UMP
a
product in aqueous solution (∼32) by measuring the
temperature dependence of the rate constant for nonenzymatic
the strengths of these hydrogen bonds as the proton affinity of
12
15−19
exchange in a model compound. Richard and Amyes used the
O4 increases.
Houk and co-workers noted this environ-
value of kex for the OMPDC-catalyzed exchange of H6 to
ment could stabilize the charge-delocalized anionic intermedi-
20
calculate an upper limit on the value of the pK of H6 of UMP
ate. However, the importance of these hydrogen bonding
interactions in the OMPDC-catalyzed reactions has not been
investigated.
a
3
in the active site (∼22). The difference (∼10 pK units)
provides a lower limit on the stabilization of the intermediate
by its interactions with the active site (≥14 kcal/mol).
a
Also, Wolfenden and Lewis recently reported that the rate of
On the basis of the structure of ScOMPDC in a complex
with 1-(5′-phospho-β-D-ribofuranosyl)barbiturate (BMP; alter-
natively, 6-hydroxyUMP), Wolfenden, Short, and their co-
workers proposed that Lys 93 (Lys 72 in MtOMPDC) is
positioned both to provide electrostatic stabilization of the
decarboxylation of 1-methylorotate, a model of OMP, is
21
increased in solvents less polar than water. At the extreme,
the rate enhancement was reported to be 6100-fold in
tetrahydrofuran, dioxane, or acetone. The slower reaction in
water is attributed to more effective solvation of the anionic
carboxylate group in the reactant than in the anionic transition
state/vinyl carbanion intermediate. These effects are similar to
(
localized) negative charge at C6 and to protonate the
6
intermediate to form the UMP product. This proposal was
4
reiterated by Cleland and co-workers, who concluded that “the
a rate enhancement of >10 reported by Lienhard and co-
13
mechanism of this enzyme is fairly well understood”.
workers for the decarboxylation of 2-(1-carboxy-1-
hydroxyethyl)thiamin pyrophosphate to yield 2-(1-
hydroxyethyl)thiamin pyrophosphate performed in dimethyl
However, in 1997, before the availability of a structure for
any OMPDC, Houk and Lee proposed that the negative charge
of the “vinyl carbanion” intermediate is not localized to C6 as
implied in the proposed role of Lys 72 (MtOMPDC) as an
electrostatic catalyst but can be resonance delocalized to O4 to
generate a “carbene” at C6 (C6 is neutral with an electron pair
2
2,23
sulfoxide instead of water.
Again, these effects were
attributed to the diminished level of solvation of the reactant
in the nonpolar solvents relative to that of the transition state.
Although not noted by Wolfenden and Lewis, the active sites
of all OMPDCs contain a hydrophobic cavity proximal to the
presumed location for the carboxylate group of the bound
OMP substrate. A reasonable hypothesis is that this hydro-
phobic cavity prevents solvation of the carboxylate group;
because the negative charge of the anionic intermediate is
2
14
in the sp orbital). Furthermore, protonation of O4 by an
active site acid was predicted to provide substantial stabilization
of the carbene intermediate. However, the structures for
divergent OMPDCs reveal the absence of an acidic functional
group proximal to O4; instead, on the basis of structures of
8
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Table 2. Data Collection and Refinement Statistics for BMP Complexes of MtOMPDC mutants
S127A
I96S
I96T
L123N
L123S
V155D
P21
V155S
Data Collection
space group
P21
P21
P21
P21
P21
P21
no. of molecules in the
asymmetric unit
2
2
2
2
2
2
2
cell dimensions
a (Å)
59.89
64.16
61.57
115.60
1.32
59.93
64.34
61.58
115.86
1.53
59.97
59.92
63.98
61.75
115.56
1.42
59.85
63.93
61.74
115.57
1.31
59.74
64.33
61.55
115.53
1.42
59.71
64.28
61.33
115.54
1.32
b (Å)
63.84
c (Å)
61.58
β (deg)
115.54
1.42
resolution (Å)
no. of unique reflections
Rmerge
98290
0.071
99.8
63155
0.087
99.4
76659
0.063
78628
0.079
99.3
95704
0.089
95.5
77594
0.058
97.9
97409
0.091
99.26
completeness (%)
97.7
Refinement
25.0−1.42
0.164
resolution (Å)
Rcryst
25.0−1.32
0.172
25.0−1.53
0.161
25.0−1.42
0.180
25.0−1.31
0.165
25.0−1.42
0.174
25−1.32
0.164
Rfree
0.193
0.186
0.185
0.205
0.177
0.202
0.177
no. of atoms
protein
3422
424
BMP
44
3388
358
BMP
44
3364
393
BMP
44
3388
432
BMP
44
3414
468
BMP
44
3404
387
BMP
44
3386
465
BMP
44
water
bound ligand
no. of ligand atoms
rmsd
bond lengths (Å)
bond angles (deg)
PDB entry
0.006
1.05
0.006
1.08
0.006
1.09
0.006
1.10
0.006
1.11
0.006
1.07
0.006
1.11
3SIZ
3NQC
3NQD
3NQE
3NQF
3NQG
3NQM
Figure 1. Representative electron density map for the active site of the S127P mutant in a complex with azaUMP and countered at 1.5σ. This figure
34
was produced with PyMOL. The details of the interactions between 6-azaUMP and the active site are described in the text.
delocalized to O4, the hydrophobic cavity is not expected to
influence solvation of the transition state/intermediate.
However, the presence of this hydrophobic cluster has not
been previously noted, so its potential importance in substrate
destabilization has not been tested.
In this work, we report the effects of site-directed
substitutions for residues in the MtOMPDC that form the
hydrophobic pocket proximal to the C6 carboxylate group as
well as for Ser 127 that hydrogen bonds to O4 of the substrate/
vinyl carbanion intermediate via its backbone amide. Our
results provide evidence that contradicts the proposal that “the
13
mechanism of this enzyme is fairly well understood”.
MATERIALS AND METHODS
Materials. azaUMP was purchased from R. I. Chemicals
Inc. Barbiturate was purchased from Sigma. All solutions were
■
8
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Biochemistry
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prepared with Millipore Ultrapure filtered water. OMP, FOMP,
FUMP, and BMP were prepared by published proce-
dures.
Seven different crystal forms (Table 2) of mutants of
MtOMPDC liganded with BMP were grown by the hanging
drop method at room temperature: (1) S127A·BMP, (2)
I96S·BMP, (3) I96T·BMP, (4) L123N·BMP, (5) L123S·BMP,
(6) V155D·BMP, and (7) V155S·BMP. The protein solutions
contained the mutant protein (30 mg/mL) in 20 mM Hepes
(pH 7.5), 150 mM NaCl, 3 mM DTT, and 40 mM BMP. The
precipitants were as follows. (1) For S127A·BMP, the
precipitant contained 30% PEG 4000, 0.1 M sodium citrate
(pH 5.6), and 0.2 M ammonium acetate. (2) For I96S·BMP,
the precipitant contained 60% tacsimate (pH 7.0). (3) For
I96T·BMP, the precipitant contained 25% PEG 3350, 0.1 M
4
,13,24,25
Cloning, Site-Directed Mutagenesis, and Protein
Expression and Purification. The gene encoding
MtOMPDC was previously cloned from M. thermoautotrophicus
strain Delta H genomic DNA into the pET-15b vector
4
,24
(
Novagen).
This plasmid served as the template for site-
directed mutagenesis by the overlap extension procedure.
Primers with the desired mutations (Eurofins MWG Operon)
were combined with external primers to generate two gene
fragments. The polymerase chain reaction product was digested
by NdeI and BamHI (Promega) followed by ligation into pET-
Bis-Tris (pH 6.5), and 0.2 M MgCl . (4) For L123N·BMP, the
2
1
5b. DNA sequencing at the University of Illinois Core
precipitant contained 30% PEG ME 2000 and 0.1 M potassium
Sequencing Facility confirmed the desired sequences. The
thiocyanate. (5) For L123S·BMP, the precipitant contained
−
mutants were purified from a kanamycin-resistant pyrF strain
25% PEG 3350, 0.1 M Bis-Tris (pH 6.5), and 0.2 M MgCl . (6)
2
of E. coli generated using Wanner’s method for chromosomal
For V155D·BMP, the precipitant contained 1.4 M sodium
citrate and 0.1 M Hepes (pH 7.5). (7) For V155S·BMP, the
precipitant contained 2.4 M sodium malonate (pH 7.0).
Prior to data collection, the crystals were transferred to
cryoprotectant solutions composed of their mother liquor and
20% glycerol and flash-cooled in a nitrogen stream. All X-ray
diffraction data sets (Tables 1 and 2) were collected at the
NSLS X4A beamline (Brookhaven National Laboratory) on an
ADSC CCD detector. Diffraction intensities were integrated
2
6
disruptions; a chloramphenicol-resistant auxiliary plasmid,
pTara, a gift from J. Cronan (University of Illinois), supplied
the T7 RNA polymerase for transcription from pET-15b. The
4,24
mutant proteins were isolated by published procedures.
Enzymatic Assays. All assays were performed at 25 °C in
1
0 mM MOPS (pH 7.1) containing 100 mM NaCl following
4,24
published procedures. Because the values of K for the wild
M
type and most mutants are <5 μM, they cannot be quantitated
2
7
by measuring the initial velocity as a function of initial substrate
and scaled with DENZO and SCALEPACK. The data
collection statistics are listed in Tables 1 and 2.
24
concentration. Instead, as described previously, the values of
k /K were determined from the first-order decay of the
absorbance when the concentration of the remaining substrate
Structure Determination and Model Refinement. All
15 structures (Tables 1 and 2) were determined by molecular
replacement with the fully automated molecular replacement
cat
M
was ≤20% of the estimated K value; the values of k were
M
cat
28
determined in separate assays with substrate concentrations
pipeline BALBES, using only input diffraction and sequence
data. Partially refined structures of all 15 crystal forms (Tables 1
and 2) were the outputs from BALBES without any manual
intervention. Subsequently, several iterative cycles of refine-
ment were performed for each crystal form, including model
exceeding the estimated values of K by a factor of ≥10. The
M
kinetic constants for mutants with values of K values of >20
M
μM were measured by initial velocity measurements.
Exchange of H6 of FUMP. Exchange of the H6 proton of
2
9
30
rebuilding with COOT, refinement with PHENIX, and
FUMP (5 mM) was measured in 100 mM glycylglycine (pD
31
1
19
automatic model rebuilding with ARP.
9
.35, I = 0.1 M with NaCl) using H and F NMR
3,4
In each structure, the polypeptides are arranged as dimers
with the monomers connected by a noncrystallographic 2-fold
axis. The active site loops, chain segment 181−189, are well-
defined in each structure. Each structure also has well-ordered
density for the inhibitor in each active site. No residues (other
than Gly) lie in disallowed regions of the Ramachandran plots
in the various structures.
Representative electron density for the S127P mutant
liganded with azaUMP is shown in Figure 1.
Final crystallographic refinement statistics for all determined
OMPDC structures are listed in Tables 1 and 2.
spectroscopies as described previously.
Crystallization and Data Collection. Eight different
crystal forms (Table 1) of mutants of MtOMPDC liganded
with azaUMP were grown by the hanging drop method at room
temperature: (1) S127G·azaUMP, (2) S127A·azaUMP, (3)
S127P·azaUMP, (4) I96S·azaUMP, (5) I96T·azaUMP, (6)
L123N·azaUMP, (7) L123S·azaUMP, and (8) V155S·azaUMP.
The protein solutions contained the mutant protein (30 mg/
mL) in 20 mM Hepes (pH 7.5), 150 mM NaCl, 3 mM DTT,
and 40 mM azaUMP. The precipitants were as follows. (1) For
S127G·azaUMP, the precipitant contained 20% PEG 3350 and
0
.15 M DL-malate (pH 7.0). (2) For S127A·azaUMP, the
RESULTS AND DISCUSSION
As summarized in the introductory section, the value of ΔG
precipitant contained 30% PEG 400, 0.1 M Hepes (pH 7.5),
and 0.2 M sodium chloride. (3) For S127P·azaUMP, the
precipitant contained 60% tacsimate (pH 7.0). (4) For
I96S·azaUMP, the precipitant contained 30% PEG 4000, 0.1
M sodium citrate (pH 5.6), and 0.2 M ammonium acetate. (5)
For I96T·azaUMP, the precipitant contained 30% PEG 4000,
■
⧧
for decarboxylation of OMP is reduced by 23 kcal/mol in the
active sites of homologous, but divergent, OMPDCs, although
the structural strategies that lead to this reduction remain
uncertain. In this work, we describe studies of two features of
the active site of MtOMPDC that have not been previously
investigated: (1) a hydrophobic pocket proximal to C6 and (2)
Ser 127 that interacts with N3 and O4 of the orotate moiety.
On the basis of the experiments described in this work, we
0
(
3
.1 M sodium citrate (pH 5.6), and 0.2 M ammonium acetate.
6) For L123N·azaUMP, the precipitant contained 25% PEG
350, 0.1 M Bis-Tris (pH 5.5), and 0.2 M lithium sulfate. (7)
For L123S·azaUMP, the precipitant contained 20% PEG 8000,
.1 M sodium cacodylate (pH 6.5), and 0.2 M magnesium
⧧
0
conclude that both features contribute to the reduction in ΔG .
acetate. (8) For V155S·azaUMP, the precipitant contained 30%
PEG ME 5000, 0.1 M MES (pH 6.5), and 0.2 M ammonium
sulfate.
Importance of the Hydrophobic Pocket. We previously
reported structures of wild-type MtOMPDC liganded with
BMP and azaUMP, analogues of the transition state/carbanion
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N-terminus of the active site loop: we recently reported that the
interaction of the sequence-proximal Val 182 in the loop with
the (β/α) -barrel scaffold favors formation of the closed, active
8
32
conformation of the enzyme; therefore, we were concerned
that polar substitutions of Pro 180 could influence the kinetic
constants by perturbing the distribution between the open and
closed conformations of the enzyme.
Structures of the Mutants with Polar Substitutions for
Hydrophobic Pocket Residues. We determined structures
for all six hydrophobic pocket mutants in the presence of BMP
(
Figures 3 and 4); we also determined structures for five of the
mutants (except V155D) in the presence of azaUMP (Figures 5
and 6). In all of the structures, the side chains of the active site
residues, including Asp 70, Lys 72, and Asp 75 (from the
adjacent polypeptide in the dimer), superimpose well with
those of the BMP- and azaUMP-liganded structures of wild-
type MtOMPDC (Figures 3 and 5), providing evidence that the
interactions of the transition state/vinyl carbanion intermediate
with the active site are preserved in each mutant. Therefore, in
our interpretation of their kinetic constants, we assume that
changes in kcat arise from the altered environment provided by
the now polar cavity adjacent to the carboxylate group of OMP.
In five of the six BMP-liganded mutant structures (I96S,
I96T, L123N, L123S, and V155S), two “conserved” water
molecules that hydrogen bond to O4 and O6 of BMP undergo
small changes in position (Figures 3 and 4). The water
proximal to O4 is conserved in all structurally characterized
OMPDCs and may participate in stabilization of the negative
charge that is localized on O4 of the vinyl carbanion/carbene
intermediate; the water proximal to O6 of BMP likely is not
present when the OMP substrate binds (recall that the complex
4
Figure 2. Active site of MtOMPDC liganded with H OMP with the
2
residues that form the hydrophobic pocket represented with space-
filling atoms.
Figure 3. Superposition of the active sites of the hydrophobic pocket
mutants liganded with BMP. The red spheres represent water
molecules.
of wild-type MtOMPDC with H OMP does not contain any
2
water molecules). The active site of the BMP-liganded L123S
contains a third (new) water that is stabilized by its proximity
to the waters adjacent to O4 and O6, the O6 oxyanion of BMP,
and the hydroxyl group of the L123S side chain.
Wild-type MtOMPDC binds azaUMP in the syn conforma-
tion, in which O2 is hydrogen bonded to the carboxamide NH2
group of Gln 185 and N3 is hydrogen bonded to the OH group
of Ser 127. However, in the mutants, azaUMP is bound in the
anti conformation in which the pyrimidine ring is rotated by
intermediate, as well as with 5,6-dihydroOMP (H OMP), a
2
stable analogue of OMP that may be a mimic of substrate
4
,32
destabilization.
The structures of the orotate binding
pockets are superimposable, although the active site of the
complex with BMP contains an “extra” water molecule
hydrogen-bonded to O6 of the 6-OH uracil (barbiturate) ring.
The active site of the previously reported complex of wild-
1
80° around the glycosidic bond (Figure 6). With this
type MtOMPDC with H OMP is shown in Figure 2. The C6
geometry and in each structure, O2 is hydrogen bonded to a
new water molecule that is not observed in the wild-type
azaUMP complex (analogous to the conserved water molecules
proximal to O6 in the BMP-liganded structures). The
substitutions for Val 155 and Ile 96 perturb the position of
the Leu 123 side chain, thereby altering the shape of the pocket.
Also, in the L123N and V155S mutants, a second new water is
hydrogen bonded to N3 of the 6-azauracil moiety.
2
carboxylate group “points” into the hydrophobic cavity that is
formed by the side chains of Ile 96, Leu 123, Val 155, and Pro
1
80. No water molecules are observed in the hydrophobic
cavity. Although these could be brought into the active site by
hydrogen bonding interactions with the carboxylate group, the
hydrophobic side cavity would prevent completion of their
preferred tetrahedral hydrogen bonding interactions, thereby
making their presence in the active site unfavorable.
Kinetic Constants for the Mutants with Polar
Substitutions for Hydrophobic Pocket Residues. For
wild-type MtOMPDC, decarboxylation of OMP is fully rate-
determining for k , although k /K has a small inverse
We constructed polar substitutions for Ile 96, Leu 123, and
Val 155, i.e., I96S, I96T, L123N, L123S, V155D, and V155S, to
increase the polarity of the active site cavity; these “targets” for
study were based on the report from Wolfenden and Lewis that
the level of nonenzymatic decarboxylation of OMP analogues is
increased as the solvent polarity is decreased because the
negative charge of the departing carboxylate group is not
cat
cat
M
dependence on viscosity, indicating that both chemical and
24
physical steps determine its value. Therefore, changes in kcat
⧧
report on changes in ΔG for decarboxylation. We also
previously demonstrated that substitutions for Asp 70 decrease
2
1
stabilized. Therefore, we hypothesized that introduction of
polar residues into this cavity (and possible “new” water
molecules that can interact with hydrogen bond donors and
the value of kcat but do not alter the value of k (exchange of
ex
H6 of the UMP product or the FUMP product analogue with
solvent), thereby providing support for the occurrence of the
vinyl carbanion intermediate (obtained either from decarbox-
ylation of OMP or from exchange of H6 of the UMP product)
acceptors) would decrease the value of k . We did not
cat
introduce substitutions for Pro 180 because it is located at the
8
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Biochemistry
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Figure 4. Active sites of the hydrophobic pocket mutants liganded with BMP. The red spheres represent water molecules. All of the active sites
contain two spatially conserved water molecules; the active site of L123S contains an additional water molecule hydrogen bonded to the OH group
of Ser 123.
4
on the reaction coordinate. We interpreted the effects of the
substitutions for Asp 70 as evidence of substrate destabilization
during the transition from the open, inactive conformation to
the closed, active conformation, presumably by unfavorable
electrostatic (and steric) interactions with the substrate
carboxylate group.
the conformational equilibrium between the open, inactive and
closed, active conformations.
32
However, for Ile 96 and Val 155 (including the V155D
mutation for which the value of kcat is substantially decreased),
the values of kex are not significantly altered by the
substitutions. The simplest interpretation is that the stability
of the anionic intermediate is not affected by the substitutions.
We measured the effects of the various polar substitutions for
the hydrophobic pocket residues on the values of (1) kcat and
K for decarboxylation of OMP and (2) k for exchange of H6
of the more reactive product analogue 5-fluoroUMP; these are
presented in Table 3.
⧧
The fact that these substitutions increase ΔG for decarbox-
M
ex
ylation but not exchange suggests that the hydrophobic pocket
⧧
contributes to the reduction in ΔG by placing the substrate
carboxylate in a destabilizing, hydrophobic environment
The values of kcat are decreased from 6- to 400-fold for the
various neutral polar substitutions; for the V155D substitution,
the value of kcat is decreased by ∼10000-fold. However, the
(
“hydrophobic stress”).
For L123N and L123S, the values of k are decreased more
ex
than the values of k . A possible explanation is provided by the
values of K for the mutants are increased <3-fold, including
cat
M
structures of azaUMP complexes of these mutants in which an
extra water is located proximal to the pyrimidine moiety in each
active site, providing the potential to influence either the rate of
that for the V155D mutant. Thus, each of the polar
substitutions decreases the rate at which the vinyl carbanion
intermediate is formed by decarboxylation but does not alter
8
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Biochemistry
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absorbance at low substrate concentrations because of the very
slow rates of reaction).
The values of kex for the D70N/I96S and D70N/V155S
double mutants are each less than that for the D70N mutant
(
by a factor of ∼8) but are significantly greater than those that
would be expected if the effects of the individual mutants were
additive (by 22- and 44-fold, respectively). Although not
“
perfect”, these data support the suggestion that the increases in
⧧
ΔG for exchange of H6 are not additive; i.e., the effects of the
hydrophobic pocket and D70N make independent contribu-
tions to substrate destabilization with neither contributing
significantly to intermediate/transition state destabilization.
Like the L123S single mutant, the D70N/L123S double
mutant shows “anomalous” behavior: the value of kcat is 10-fold
⧧
lower than the value predicted if the effects on ΔG were
additive; however, the value of k is reduced 1500-fold from
ex
that observed for wild-type MtOMPDC and 300-fold from
those observed for D70N/I96S and D70N/V155S double
mutants. Again, these lower than expected values for kcat and kex
may result from the extra water molecule proximal to the
pyrimidine moiety.
Thus, we conclude that the reduction in ΔG by
MtOMPDC involves at least two structurally independent
mechanisms to achieve substrate destabilization.
Figure 5. Superposition of the active sites of the hydrophobic pocket
mutants liganded with azaUMP. The red spheres represent water
molecules.
formation of the anion intermediate by proton abstraction or
the stability of the intermediate.
⧧
Thus, for the neutral polar substitutions, the decreases in kcat
support the hypothesis that enhanced polarity proximal to C6
of the orotate substrate decreases the rate of decarboxylation by
stabilizing the anionic carboxylate group relative to the anionic
intermediate, as suggested by Wolfenden and Lewis to explain
the enhanced rate of decarboxylation in solvents less polar than
water. The very substantial effect of the V155D substitution
likely can be best explained by unfavorable electrostatic
interactions with the substrate carboxylate group as the
reaction coordinate for decarboxylation is traversed.
Importance of Ser 127 in Stabilization of an Anionic
Transition State/Intermediate. As proposed by Houk and
14,20
co-workers,
a hydrogen bond donor adjacent to O4 of the
OMP substrate could contribute significantly to the reduction
⧧
in ΔG by stabilization of the transition state/anionic
intermediate as developing negative charge in the pyrimidine
moiety is delocalized to O4. Structural studies of divergent
OMPDCs reveal that O4 of the substrate always is hydrogen
bonded to the backbone NH group of a conserved Ser (or,
occasionally, Thr) in the loop following the fifth β-strand of the
We note that a hydrophobic pocket proximal to the substrate
carboxylate group is conserved in all OMPDCs, despite
considerable divergence in sequence, so all OMPDCs
presumably adopt this structural strategy for reducing the
(
β/α) -barrel domain (Ser 127 in MtOMPDC) as well as the
8
spatially conserved water molecule. In addition, the side chain
OH group of the Ser residue is hydrogen bonded to the proton
on N3. The quantitative importance of these interactions in the
⧧
value of ΔG .
Independent Mechanisms for Ground State Destabi-
lization? As summarized in the introductory section, we
recently reported that the D70N and D70G substitutions also
decrease the value of kcat without influencing the value of kex;
these effects were interpreted as evidence of ground state
destabilization of the substrate’s carboxylate by its proximity to
⧧
reduction of ΔG has not been reported.
We constructed and kinetically characterized the S127A,
S127G, and S127P mutants of MtOMPDC to obtain
information about the importance of these interactions; we
also obtained structures of the S127A, S127G, and S127P
mutants liganded with azaUMP. The S127A and S127G
substitutions allow the importance of the interactions between
the side chain OH group and N3 of the orotate moiety to be
investigated. The S127P substitution replaces the backbone NH
group with Cγ of the side chain; although we recognized that
this likely would introduce a steric clash with O4 of the OMP
substrate, we considered this mutant to be worth characterizing.
Structures of the S127A, S127G, and S127P Mutants.
We determined structures for the Ala, Gly, and Pro
substitutions for Ser 127 in the presence of azaUMP. The
structures of the active sites of the mutants superimpose well
with that of wild-type MtOMPDC in a complex with azaUMP
(Figure 7), with small deviations in backbone geometry
observed for residue 127 and the two flanking residues (Met
126 and His 128). In the structure of the S127G mutant, a
water molecule occupies the position of the OH group of the
Ser residue in wild-type MtOMPDC and is hydrogen bonded
to both N3 of the pyrimidine ring and the carboxamide group
of Gln 185; this water molecule is not present in S127A,
presumably because of steric exclusion. The positions of the
4
the anionic carboxylate group of Asp 70. We now have
concluded that the hydrophobic pocket prevents stabilization of
the substrate carboxylate group relative to the transition state/
vinyl carbanion intermediate. Therefore, we constructed the
D70N/I96S, D70N/L123S, and D70N/V155S double mutants
in an attempt to obtain evidence that these structurally distinct
effects make independent contributions to the reduction in
⧧
ΔG .
The values of k , K , and k for the double mutants also are
cat
M
ex
listed in Table 3. The values of kcat for the D70N, V155S, and
I96S single mutants are reduced by factors of 200−400-fold.
For the double mutants with these residues, the values of k
cat
are reduced by factors that approximate the reductions
⧧
observed for the single mutants; i.e., the increases in ΔG are
additive, supporting the conclusion that electrostatic repulsion
by Asp 70 and hydrophobic stress by the hydrophobic pocket
independently contribute to substrate destabilization as the
transition state for decarboxylation is achieved (we could not
measure the values of K by analysis of the first-order decay in
M
8
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Biochemistry
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Figure 6. Active sites of the hydrophobic pocket mutants liganded with azaUMP. The red spheres represent water molecules. All of the active sites
contain two spatially conserved water molecules; the active sites of L123S and V155S contain an additional water molecule hydrogen bonded to the
OH group of Ser 123.
Table 3. Kinetics of Hydrophobic Pocket Mutants
−
1
k /K (M−1 s )
−1
kex (s−1)
0.015 ± 0.004
OMPDC
kcat (s )
KM (μM)
cat
M
wild type
I96T
4.0 ± 0.2
0.69 ± 0.05
0.011 ± 0.002
0.64 ± 0.01
0.36 ± 0.04
1.6 ± 0.1
2.3 ± 0.06
1.0 ± 0.04
3.5 ± 0.2
3.0 ± 0.5
4.7 ± 0.1
4.6 ± 0.5
6.0 ± 0.3
2.0 × 106
3.0 × 105
1.0 × 104
0.0047 ± 0.0004
0.006 ± 0.001
0.0007 ± 0.0001
0.00039 ± 0.00001
0.006 ± 0.003
0.057 ± 0.002
0.005 ± 0.002
0.002 ± 0.0003 (0.13) (22)
0.00001 ± 0.000007 (1500) (1.7)
0.0019 ± 0.0006 (0.13) (44)
I96S
5
L123N
1.9 × 10
L123S
1.2 × 105
2.2 × 104
V155S
0.011 ± 0.003
(5 ± 2) × 10−
0.024 ± 0.05
4
2
V155D
4.6 × 10
D70N
4.0 × 103
−
5
a
d
a
D70N/I96S
D70N/L123S
D70N/V155S
(6.0 ± 1.0) × 10 (5)
−
−
−
−
(9.3 ± 0.1) × 10− (0.1)
4
b
c
−
−
e
b
(1.8 ± 0.4) × 10− (1.5)
4
f
c
a
b
c
d
e
(
D70N/I96S)/(D70N)(I96S). (D70N/L123S)/(D70N)(L123S). (D70N/V155S)/(D70N)(V155S). (D70N/I96S)/(D70N). (D70N/
L123S)/(D70N). (D70N/V155S)/(D70N).
f
8
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presence of a water molecule that occupies the position of the
Ser OH group, thereby mimicking the OH group.
The values of kcat and K cannot be measured for the S127P
M
mutant; the value of k /KM is determined from the linear
cat
dependence of velocity on substrate concentration. The value
6
so determined is reduced by an impressive factor of 2.5 × 10 .
Quantitative interpretation of this effect in the context of the
interaction between O4 and the backbone NH group of Ser
1
27 is equivocal. The structure reveals, as expected, that the Pro
substitution alters the position of the pyrimidine ring in the
active site: O4 of the ligand is displaced 1.1 Å from the position
of O4 in the wild type and 1.5 Å from O4 in the S127A and
S127G mutants; it obviously cannot form a hydrogen bond
with the backbone. However, the distance between N6
(
homologue of C6 in the vinyl anion/carbene) and Nε of
Figure 7. Superposition of the active sites of the mutants of Ser 127
liganded with azaUMP. The red spheres represent water molecules.
Lys 72 is decreased by 0.3 Å in the mutant (2.7 Å) relative to
that in the wild type (3.0 Å). If electrostatic effects between Lys
7
2 and the anionic intermediate are of primary importance in
Table 4. Kinetics of Ser 127 Mutants
⧧
reducing the value of ΔG , the decrease in the distance in the
S127P mutant would be expected to have an only “minor”
effect on kcat (23% assuming Coulomb’s law applies to the
interaction between the ε-ammonium group of Lys 72 and the
anionic C6, i.e., no charge delocalization to form a carbene in
the anionic intermediate). Thus, the very large reduction in the
value of kcat observed for the S127P mutant is an indication that
the interaction of O4 with the backbone NH group of Ser 127
contributes significantly to stabilization of the vinyl anion/
carbene intermediate.
Houk and Lee proposed an anionic carbene resonance
structure for the anionic intermediate in which negative charge
is delocalized to O4.
that the strength of the hydrogen bond to the backbone NH
group of Ser 127 increases as negative charge is delocalized to
O4, we find this interaction could provide significant
stabilization of the intermediate and contribute to the reduction
in ΔG . However, quantitation of this stabilization is difficult
because (1) removal of the O4 hydrogen bond acceptor, i.e., 4-
deoxyuracil, will also result in the loss of a proton from N3,
thereby eliminating the hydrogen bond to Oβ of Ser 127, and
(2) alteration of the hydrogen bond donor (the NH group of
Ser 127) cannot be readily accomplished, even with unnatural
mutagenesis (in principle, replacement of the peptide bond
linkage with an ester linkage may be possible, but in practice,
ribosome-mediated synthesis of depsipeptide linkages in
proteins is difficult).
−
1
k /K (M−1 s )
−1
OMPDC
kcat (s )
KM (μM)
cat M
wild type
S127A
4.6 ± 0.4
0.035 ± 0.002
0.26 ± 0.007
2.1 ± 0.1
96 ± 5
59 ± 5
−
(2.2 ± 0.2) × 106
(3.6 ± 0.3) × 102
3
S127G
S127P
(4.3 ± 0.3) × 10
a
−
0.88 ± 0.16
a
Velocity is directly proportional to substrate concentration.
side chains of the active site residues, including Asp 70, Lys 72,
and Asp 75 (from the adjacent polypeptide), are not affected by
the substitutions for Ser 127.
The positions of the ligands in the various azaUMP-liganded
complexes are shifted from the positions in wild-type
MtOMPDC, with the effects most pronounced for the 6-
azauracil moiety (Figures 7 and 8). For example, in the S127P
mutant, O4 is displaced by 1.1 Å from the position of O4 in the
wild type and by 1.5 Å from the position of O4 in the S127A
and S127G mutants because of the steric clash with the
aliphatic Pro ring; also, N6 is positioned 0.6 Å closer to the
carboxylate group of Asp 70 and 0.3 Å closer to the ε-
ammonium group of Lys 72 than it is in the wild type. Although
these changes complicate precise structure-based interpretation
of the kinetic constants (vide infra), we decided that
characterization of this substitution would provide relevant
information about the role of Ser 127.
Kinetic Constants for the S127A, S127G, and S127P
Mutants. The S127A and S127G mutants retain the
interaction between O4 of the pyrimidine ring and the
backbone NH of residue 127, although these alter the
interactions between the side chain and N3 of the pyrimidine
ring as well as the carboxamide group of Gln 185.
14,20
Making the reasonable assumption
⧧
17
Conclusions. The structural strategies by which the
remarkable rate enhancement achieved by OMPDCs (k /
cat
1
6
knon = 7.1 × 10 ) are not well-defined. Although electrostatic
stabilization of the anionic intermediate by Lys 72 is frequently
used to explain the rate acceleration, we are investigating the
role of other active site residues. We previously demonstrated
that Asp 70 contributes modestly to the rate enhancement by
destabilization of the substrate. We now conclude that a
hydrophobic cavity proximal to the carboxylate group of the
substrate also destabilizes the substrate, presumably by
preventing the carboxylate group from being stabilized by
waters of hydration when it is bound in the active site. This
effect is also modest but likely is employed by all OMPDCs
given that residues in the hydrophobic cavity are conserved.
The importance of hydrogen bonding interactions with O4 of
the pyrimidine moiety of the substrate is more difficult to
investigate experimentally because the hydrogen bond donor is
a peptide backbone NH group of Ser 127. Replacement of Ser
For both mutants, the values of K are increased (Table 4),
M
consistent with the proposal that the hydrogen bond between
the OH group of Ser 127 and the carboxamide group of Gln
1
85 provides a “clamp” for stabilizing the closed, active
33
conformation of the enzyme. The value of kcat is reduced
130-fold for the S127A mutant and ∼18-fold for the S127G
∼
mutant. The reduction for the Ala substitution suggests that a
hydrogen bond between the OH group of Ser 127 and the
proton of N3 provides stabilization of the enhanced,
delocalized anionic charge in the pyrimidine ring that occurs
as the vinyl anion/carbene intermediate is formed. The partial
“
rescue” observed for the Gly substitution is explained by the
8
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Figure 8. Active sites of the mutants of Ser 127 liganded with azaUMP. The red spheres represent water molecules. Distances for important
interactions of the ligand with active site residues are shown.
1
27 with proline (S127P) has a large effect on k /K (a
AUTHOR INFORMATION
Corresponding Author
*Institute for Genomic Biology, University of Illinois, 1206 W.
Gregory Dr., Urbana, IL 61801. Phone: (217) 244-7414. Fax:
cat
M
■
6
reduction of 2.5 × 10 -fold), supporting the proposal by Lee
and Houk that the hydrogen bond between O4 of the substrate
and the backbone NH is important in reducing ΔG .
⧧
(
217) 333-0508. E-mail: j-gerlt@uiuc.edu.
Funding
ASSOCIATED CONTENT
■
†
This research was supported by a National Institutes of Health
Accession Codes
(
NIH) Chemistry Biology Interface Training Grant under Ruth
The X-ray coordinates and structure factors for the following
structures have been deposited in the Protein Data Bank: the
S127G mutant of MtOMPDC in the presence of azaUMP
L. Kirschstein National Research Service Award
T32GM070241 (to V.I.) and NIH Grant GM065155 (to
J.A.G.).
(
entry 3LLD), the S127A mutant of MtOMPDC in the
ACKNOWLEDGMENTS
■
presence of azaUMP (entry 3SY5), the S127A mutant of
MtOMPDC in the presence of BMP (entry 3SIZ), the S127P
mutant of MtOMPDC in the presence of azaUMP (entry
Molecular graphics images were produced using the UCSF
Chimera package from the Resource for Biocomputing,
Visualization, and Informatics at the University of California,
San Francisco (supported by NIH Grant P41 RR-01081).
3
LLF), the I96S mutant of MtOMPDC in the presence of
azaUMP (entry 3PBU), the I96S mutant of MtOMPDC in the
presence of BMP (entry 3NQC), the I96T mutant of
MtOMPDC in the presence of azaUMP (entry 3RPV), the
I96T mutant of MtOMPDC in the presence of BMP (entry
ABBREVIATIONS
■
FOMP, 5-fluoroorotidine 5′-monophosphate; FUMP, 5-fluo-
roUMP; azaUMP, 6-azauridine 5′-monophosphate; BMP, 1-(5′-
phospho-β-D-ribofuranosyl)barbituric acid; MtOMPDC,
OMPDC from M. thermoautotrophicus; OMP, orotidine 5′-
monophosphate; OMPDC, orotidine 5′-monophosphate de-
carboxylase; rmsd, root-mean-square deviation; ScOMPDC,
OMPDC from S. cerevisiae; WT, wild type.
3
NQD), the L123N mutant of MtOMPDC in the presence of
azaUMP (entry 3PBW), the L123N mutant of MtOMPDC in
the presence of BMP (entry 3NQE), the L123S mutant of
MtOMPDC in the presence of azaUMP (entry 3PBY), the
L123S mutant of MtOMPDC in the presence of BMP (entry
REFERENCES
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NQF), the V155D mutant of MtOMPDC in the presence of
■
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Richard, J. P. (2007) Product deuterium isotope effect for orotidine 5′-
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dx.doi.org/10.1021/bi2012355|Biochemistry 2011, 50, 8497−8507

Biochemistry
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