Conformational changes in orotidine 5′-monophosphate decarboxylase: A structure-based explanation for how the 5′-phosphate group activates the enzyme

Article abstract of DOI:10.1021/bi301188k

The binding of a ligand to orotidine 5′-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (E_o) to a closed, active conformation (E_c). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate (in the E _c?S^? complex) are enforced. Focusing on the OMPDC from Methanothermobacter thermautotrophicus, we find the "remote" 5′-phosphate group of the substrate activates the enzyme 2.4 × 10⁸-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that (1) directly contact the 5′-phosphate group, (2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding interactions across the interface between the "mobile" and "fixed" half-barrel domains of the (β/α)₈-barrel structure. Our data support a model in which the IBE provided by the 5′-phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the E_c?S and E_c?S^? complexes relative to the E_o?S complex. The conclusion that the IBE of the 5′-phosphate group provides stabilization to both the E _c?S and E_c?S^? complexes, not just the E_c?S^? complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions.

Full text of DOI:10.1021/bi301188k

Article
pubs.acs.org/biochemistry
Conformational Changes in Orotidine 5′-Monophosphate
Decarboxylase: A Structure-Based Explanation for How the 5′-
Phosphate Group Activates the Enzyme
Bijoy J. Desai,† B. McKay Wood,† Alexander A. Fedorov,‡ Elena V. Fedorov,‡ Bogdana Goryanova,§
Tina L. Amyes,§ John P. Richard,§ Steven C. Almo,‡ and John A. Gerlt*,†
†Departments of Biochemistry and Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States
‡Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States
§Department of Chemistry, University at Buffalo, Buffalo, New York 14260, United States
ABSTRACT: The binding of a ligand to orotidine 5′-mono-
phosphate decarboxylase (OMPDC) is accompanied by a conforma-
tional change from an open, inactive conformation (Eo) to a closed,
active conformation (Ec). As the substrate traverses the reaction
coordinate to form the stabilized vinyl carbanion/carbene
intermediate, interactions that destabilize the carboxylate group of
the substrate and stabilize the intermediate (in the Ec·S⧧ complex)
are enforced. Focusing on the OMPDC from Methanothermobacter
thermautotrophicus, we find the “remote” 5′-phosphate group of the
substrate activates the enzyme 2.4 × 108-fold; the activation is
equivalently described by an intrinsic binding energy (IBE) of 11.4
kcal/mol. We studied residues in the activation that (1) directly
contact the 5′-phosphate group, (2) participate in a hydrophobic
cluster near the base of the active site loop that sequesters the bound substrate from the solvent, and (3) form hydrogen bonding
interactions across the interface between the “mobile” and “fixed” half-barrel domains of the (β/α)8-barrel structure. Our data
support a model in which the IBE provided by the 5′-phosphate group is used to allow interactions both near the N-terminus of
the active site loop and across the domain interface that stabilize both the Ec·S and Ec·S⧧ complexes relative to the Eo·S complex.
The conclusion that the IBE of the 5′-phosphate group provides stabilization to both the Ec·S and Ec·S⧧ complexes, not just the
Ec·S⧧ complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo
design of enzymes that catalyze novel reactions.
rotidine 5′-monophosphate decarboxylase [OMPDC
(Scheme 1)] that catalyzes the final step in pyrimidine
residues in the OMPDC from Methanothermobacter thermauto-
trophicus, MtOMPDC, the focus of this article, with BMP [l-(5-
phospho-β-ribofuranosyl)barbituric acid], an intermediate/
transition state analogue,11 are shown in Figure 1. Each active
site contains an essential Asp-Lys-Asp motif involving Asp 70
and Lys 72 from one polypeptide and Asp 75* from the
symmetry-related polypeptide as well as hydrogen bonding
interactions between the pyrimidine ring and both the OH
group and the backbone amide group of Ser 127.
O
Scheme 1
Two structural strategies can be envisioned for destabiliza-
tion of the OMP substrate as the transition state is formed: (1)
electrostatic/steric destabilization of the carboxylate group by
the proximal Asp (Asp 70)12,13 and (2) desolvation of the
carboxylate group by a proximal hydrophobic cavity (formed by
Ile 96, Leu 123, Val 155, and Pro 180).14,15 The importance of
these interactions has been tested; each contributes ∼4 kcal/
mol to the reduction in ΔG⧧.
biosynthesis is a remarkable catalyst: the rate enhancement
(kcat/knon = 7.1 × 1016) and the catalytic proficiency [(kcat/Km)/
knon = 4.8 × 10−22 M] (affinity for the transition state) are
among the largest measured for enzyme-catalyzed reactions.2,3
We, and others,4−7 have been interested in defining the
strategies used by OMPDC to reduce the value of ΔG⧧ by 23
kcal/mol. The reaction coordinate involves a vinyl carbanion/
carbene intermediate that is stabilized by ≥14 kcal/mol by its
interactions with the active site.8−10
Received: September 2, 2012
Revised: October 2, 2012
Published: October 3, 2012
OMPDCs are obligate dimers, with the active sites located at
the dimer interface. The interactions of conserved active site
© 2012 American Chemical Society
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Figure 1. Active site of MtOMPDC showing the interactions of active
site residues with BMP, an intermediate/transition state analogue.
Figure 2. Superposition of the structures of individual polypeptides of
unliganded (Eo) and BMP-liganded (Ec) MtOMPDC, showing the
movement (within the box; in the direction of the arrow) of the
“mobile” domain (red α-helices and blue β-strands) toward the “fixed”
domain (magenta α-helices and green β-strands). The ordered active
site loop is colored yellow in the BMP-liganded structure.
Also, at least two structural strategies can be envisioned for
stabilization of the anionic intermediate as the transition state is
formed: (1) attractive electrostatic interactions with the ε-
ammonium group of Lys 72 that protonates C6 of the
intermediate6 and (2) enhanced hydrogen bonding interactions
with the backbone amide group of Ser 127.16,17 For the latter
strategy to be effective, the negative charge must be delocalized
to O4 in the pyrimidine ring [as described by the carbene
resonance structure (Scheme 1)]; calculations support the
participation of this interaction, although its importance has not
been tested directly.16,17
Scheme 2
These destabilizing/stabilizing interactions are “enforced” by
a closed, catalytically active conformation (Ec) that is observed
crystallographically in the presence of the intermediate/
transition state analogues 6-azaUMP18 and BMP;11 in their
absence, MtOMPDC exists in an open, catalytically inactive
conformation (Eo) (Figure 2).12,13 The more favorable
transition from Eo to Ec in the Eo·S and Ec·S complexes [Kc
vs Kc′ in the kinetic model shown in Scheme 2 and Figure 319
(vide infra)] is responsible, in part, for a 2.4 × 108-fold
activation of the enzyme and involves (1) closure of the nine-
residue active site loop (Pro 180−Asp 188) that sequesters the
substrate from solvent and (2) formation of a more compact
conformation for the (β/α)8-barrel structure that juxtaposes the
substrate with the residues involved in substrate destabilization
and intermediate stabilization.
Each (β/α)8-barrel in the obligate dimer is formed from two
“half-barrel” domains, a “fixed” domain (including β-strands 2−
5) located at the dimer interface and a “mobile” domain
(including β-strands 6−8, and 1);20 in the transition from Eo
and Ec, the “mobile” domain moves toward the “fixed” domain,
constricting the active site cavity that envelops the substrate
Figure 3. Three-dimensional free energy diagram for the kinetic model
depicted in Scheme 2. See the text for a detailed explanation.
a
(Figure 2). Because the active site loop sequesters the
substrate from solvent in the Ec·S complex, substrate binding to
Eo must precede the conformational change. Thus, “induced
fit”, not “conformational selection”, is responsible for altering
the distribution between Eo and Ec when the substrate
defined as an association complex but Km is a dissociation
constant]} and (2) stabilizing both Ec·S and Ec·S⧧ (vide infra)
relative to Eo·S (Kc in Scheme 2). The fact that Ec is unstable
relative to Eo in the absence of substrate (Kc′) facilitates release
of the UMP product; if the total energy obtained from substrate
binding were used only to “grip” the substrate, release of the
product from the Ec·P complex likely would be rate-
determining.22 Therefore, the structural and energetic coupling
between substrate binding to Eo and the subsequent transition
b
,
binds.21
The energy obtained from formation of the Eo·S complex
from Eo and S is partitioned between (1) “gripping” the
substrate {with the value of Km providing a measure of the
affinity for the substrate [1/(KcKs) in Scheme 2, where Ks is
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Table 1. Data Collection and Refinement Statistics for BMP Complexes of Single Mutants
Q185A
R203A
T159V
Data Collection
P21 P212121
T159A
T159S
R160A
P21
Y206F
P21
K82A
space group
P21
P21212
P21
P21
no. of molecules in the
asymmetric unit
2
2
2
2
2
2
2
2
cell dimensions
a (Å)
59.80
63.86
61.47
115.28
1.37
80.00
64.00
73.30
59.98
64.12
61.89
115.46
1.40
52.61
74.04
117.50
58.67
73.87
59.52
119.50
1.3
59.80
64.14
61.78
115.67
1.4
59.62
63.41
61.04
115.02
1.42
59.78
64.01
61.63
115.52
1.49
b (Å)
c (Å)
β (deg)
resolution (Å)
no. of unique reflections
Rmerge
1.50
1.60
87599
0.065
99.7
58327
0.082
95.6
79912
0.091
95.8
54292
0.076
88.5
104965
0.038
96.8
76701
0.083
91.5
76776
0.073
98.1
68389
0.043
99.7
completeness (%)
Refinement
resolution (Å)
Rcryst
25.0−1.37
0.165
25.0−1.50
0.223
25.0−1.40
0.188
25.0−1.60
0.204
25.0−1.3
0.165
25.0−1.40
0.199
25−1.42
0.172
25−1.49
0.153
Rfree
0.178
0.247
0.206
0.231
0.176
0.222
0.193
0.174
no. of atoms
protein
3421
3412
144
3364
396
3322
3418
410
3374
410
3379
347
3416
444
water
454
170
bound ligands
BMP SO4
BMP
BMP
BMP GOL
BMP GOL,
POL
BMP
BMP GOL BMP GOL
no. of ligand atoms
rmsd
54
44
44
50
82
44
50
50
bond lengths (Å)
bond angles (deg)
PDB entry
0.006
1.0
0.005
1.2
0.006
1.1
0.007
1.1
0.006
1.1
0.006
1.1
0.006
1.1
0.006
1.1
3V1P
3LI0
3P60
3P5Y
3P5Z
3P61
3RLV
3RLU
from Eo·S to Ec·S are essential for understanding the catalytic
strategy that produces the extraordinary rate enhancement.
We recently reported initial efforts to identify structural
features that stabilize Ec·S.19 Those focused on a hydrophobic
cluster that includes Val 182 in the active site loop as well as Ile
199, Val 201, and Ile 218 located near the C-terminal ends of
the seventh and eighth β-strands in the “mobile” domain.
Substitutions of these residues that are “remote” from the
binding site for the 5′-phosphate group with Ala increase the
value of Km but have little effect on the value of kcat. The
simplest interpretation is that the hydrophobic cluster stabilizes
the closed conformation of the active site loop and, therefore,
contributes to the stability of Ec·S. Without this stabilization,
the formation of Ec·S from Eo and S is less favorable, requiring
increased concentrations of substrate to provide the energy to
allow its formation (as revealed by an increase in the value of
Km). However, when Ec·S is formed, it is fully competent to
yield product via the vinyl anion/carbene intermediate and the
transition state that precedes it on the reaction coordinate
(Ec·S⧧) so the value of kcat (the difference in energy between
Ec·S and Ec·S⧧) is unchanged.
In this work, we focus on residues involved in hydrogen
bonding interactions between the “mobile” and “fixed” domains
observed in Ec but not in Eo (involving Thr 159, Arg 160, and
Tyr 206), also “remote” from the binding site for the 5′-
phosphate group, to determine whether these contribute to the
stability of Ec·S. We also target the only two residues (Gln 185
and Arg 203) that provide direct side chain contacts with the
5′-phosphate group of the substrate and, therefore, reasonably
would be involved in generation of the intrinsic binding energy
(IBE) of the 5′-phosphate group that is used to allow
stabilization of both Ec·S and Ec·S⧧ relative to Eo and S.23
Finally, we examine double mutants involving one “remote”
residue that contributes to the stability of Ec·S (Val 182, Thr
159, or Arg 160) and one residue that generates the IBE of the
5′-phosphate group (Arg 203). The kinetic and structural
properties of the mutants are consistent with a structure-based
model in which Ec·S and Ec·S⧧ are stabilized equally relative to
Eo·S by (1) the interaction of the 5′-phosphate group with its
binding site that allows and/or directs reorganization of the
active site loop, (2) assembly of the hydrophobic cluster near
the N-terminus of the reorganized active site loop that stabilizes
its closed conformation, and (3) formation of interactions
across the domain interface in Ec·S and Ec·S⧧, some of which
(those involving Thr 159 and Arg 160) are spatially proximal to
the reorganized active site loop and, therefore, are influenced by
its structure. This analysis clarifies the role of the IBE of the 5′-
phosphate group in reducing the value of ΔG⧧ and provides the
framework for a more detailed investigation of the structural
features involved in both generation of the IBE and its use to
facilitate the conversion of Eo·S to both Ec·S and Ec·S⧧.
The conclusion that the IBE generated by interaction of the
5′-phosphate group with its binding site allows stabilization of
both the Ec·S and Ec·S⧧ complexes, not just the Ec·S⧧ complex,
relative to Eo and S by allowing formation of “remote”
interactions that stabilize Ec likely can be applied to many (all?)
enzymes in which conformational changes accompany substrate
binding.
MATERIALS AND METHODS
■
Materials. OMP, EO, FEO, and BMP were prepared by
published procedures.6,11,13,24−26 All solutions were prepared
with Millipore Ultrapure filtered water.
Protein Expression and Purification and Site-Directed
Mutagenesis. The gene encoding MtOMPDC was previously
cloned in the pET-15b vector (Novagen). The site mutants
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Table 2. Data Collection and Refinement Statistics for BMP Complexes of Double and Triple Mutants
T159V/
V182A/
Y206F
Q185A/
R203A
T159V/
V182A
T159V/
Y206F
V182A/
Y206F
R160A/
V182A
R160A/
Y206F
R203A/
V182A
R203A/
T159V
R203A/
R160A
Data Collection
space group
P41212
P21
P21
P21
P21
P21
P21
P21
P21
P212121
no. of molecules in
the asymmetric
unit
2
2
2
2
2
2
2
2
2
2
cell dimensions
a (Å)
91.51
91.51
135.38
59.77
64.07
61.64
115.46
1.54
59.93
63.67
61.43
115.34
1.34
59.56
63.62
61.26
115.00
1.32
59.77
64.07
61.64
115.46
1.32
60.08
63.83
61.91
115.53
1.26
59.81
63.98
61.43
115.48
1.32
55.08
66.40
59.55
101.85
1.53
54.35
63.06
54.51
98.95
1.71
56.62
56.61
127.21
b (Å)
c (Å)
β (deg)
resolution (Å)
1.94
1.42
no. of unique
41820
55241
87118
96995
95690
111994
97927
62002
38041
77257
reflections
Rmerge
0.095
96.8
0.075
89.4
0.065
93.0
0.055
99.8
0.088
97.4
0.073
98.4
0.058
99.8
0.047
98.0
0.092
96.1
0.065
92.2
completeness (%)
Refinement
25.0−1.32
0.231
resolution (Å)
Rcryst
25−1.94
0.172
25−1.54
0.173
25−1.34
0.173
25.0−1.32
0.165
25.0−1.26
0.176
25−1.32
0.164
25−1.53
0.146
25−1.71
0.225
25−1.4
0.184
Rfree
0.198
0.206
0.192
0.178
0.247
0.196
0.183
0.172
0.268
0.211
no. of atoms
protein
3254
3390
3477
3434
406
3327
342
3444
553
BMP
44
3426
3499
3314
74
3359
225
BMP
44
water
191
367
464
373
bound ligands
BMP SO4
BMP GOL
50
BMP GOL
50
BMP GOL
50
BMP
44
BMP GOL
50
BMP GOL
69
BMP
44
no. of ligand atoms 54
rmsd
bond
lengths (Å)
0.007
0.007
1.1
0.006
1.1
0.006
1.1
0.006
1.0
0.006
1.1
0.006
1.1
0.006
1.1
0.008
1.0
0.006
1.1
bond
angles
(deg)
1.00
PDB entry
4FX8
3QEZ
3QF0
3QMT
3QMR
3SJ3
3QMS
4FX6
4FXR
4GC4
were constructed (overlap extension), and the wild-type and
mutant proteins were expressed and prepared following
previously published procedures.13,15,24
Assay for Decarboxylation of OMP. Assays were
performed at 25 °C in 10 mM MOPS (pH 7.1) containing
100 mM NaCl following published procedures.13,15,24
Assay for Decarboxylation of EO. The values of kcat/Km
for decarboxylation of EO were determined as previously
described.15,25
Assay for Decarboxylation of FEO. For wild-type
MtOMPDC and single mutants, the values of kcat/Km for
decarboxylation of FEO were determined by quantitating the
first-order decay of FEO at 290 nm using procedures developed
for EO.15,25
For double and triple mutants, the values of kcat/Km for
decarboxylation of FEO were determined using high-perform-
ance liquid chromatography-based end point assays using
procedures developed for EO;15,25 the amount of FEU product
was quantitated using 5-fluorouridine as the reference standard
(ε = 9660 M−1 cm−1 in 0.1 N HCl).
Crystallization and Data Collection. Eight different
crystal forms (Table 1) were grown by the sitting drop method
at room temperature for single mutants of MtOMPDC
liganded with BMP: (1) Q185A·BMP, (2) R203A·BMP, (3)
T159V·BMP, (4) T159A·BMP, (5) T159S·BMP, (6)
R160A·BMP, (7) Y206F·BMP, and (8) K82A·BMP. The
protein solutions for all eight crystallizations contained the
corresponding protein (30 mg/mL) in 20 mM Hepes (pH 7.5),
100 mM NaCl, 3 mM DTT, and 40 mM BMP. The
precipitants were as follows. (1) For the Q185·BMP complex,
the precipitant contained 30% PEG 4000, 0.1 M Tris-HCl (pH
8.5), and 0.2 M lithium sulfate. (2) For the R203A·BMP
complex, the precipitant contained 2.4 M sodium malonate
(pH 7.0). (3) For the T159V·BMP complex, the precipitant
contained 30% PEG 4000, 0.1 M sodium citrate (pH 5.6), and
0.2 M ammonium sulfate. (4) For the T159A·BMP complex,
the precipitant contained 20% PEG 4000, 20% 2-propanol, and
0.1 M sodium citrate (pH 5.6). (5) For the T159S·BMP
complex, the precipitant contained 20% PEG 4000, 20% 2-
propanol, and 0.1 M sodium citrate (pH 5.6). (6) For the
R160A·BMP complex, the precipitant contained 30% PEG
4000, 0.1 M sodium citrate (pH 5.6), and 0.2 M ammonium
acetate. (7) For the Y206F·BMP complex, the precipitant
contained 30% PEG 4000, 0.1 M HEPES (pH 7.5), and 0.2 M
magnesium chloride. (8) For the K82A·BMP complex, the
precipitant contained 20% PEG 8000, 0.1 M Tris-HCl (pH
8.5), and 0.2 M magnesium chloride.
Ten different crystal forms (Tables 2) were grown by the
sitting drop method at room temperature for double and triple
mutants of MtOMPDC liganded with BMP: (1) Q185A/
R203A·BMP, (2) T159V/V182A·BMP, (3) T159V/
Y206F·BMP, (4) V182A/Y206F·BMP, (5) R160A/
V182A·BMP, (6) R160A/Y206F·BMP, (7) R203A/
V182·BMP, (8) R203A/T159V·BMP, (9) R203A/
R160A·BMP, and (10) T159V/V182A/Y206F·BMP. The
protein solutions for all 10 cocrystallizations contained the
corresponding protein (30 mg/mL) in 20 mM Hepes (pH 7.5),
100 mM NaCl, 3 mM DTT, and 40 mM BMP. The
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precipitants were as follows. (1) For the Q185A/R203A·BMP
complex, the precipitant contained 2.0 M ammonium sulfate
(pH 4.9). (2) For the T159V/V182A·BMP complex, the
precipitant contained 0.4 M sodium phosphate, 1.6 M
potassium phosphate, 0.1 M imidazole (pH 8.0), and 0.2 M
sodium chloride. (3) For the T159V/Y206F·BMP complex, the
precipitant contained 1.4 M sodium citrate and 0.1 M HEPES
(pH 7.5). (4) For the V182A/Y206F·BMP complex, the
precipitant contained 0.8 M sodium phosphate, 1.2 M
potassium phosphate, and 0.1 M acetate (pH 4.5). (5) For
the R160A/V182A·BMP complex, the precipitant contained
30% PEG 4000, 0.1 M Tris-HCl (pH 8.5), and 0.2 M lithium
sulfate. (6) For the R160A/Y206F·BMP complex, the
precipitant contained 30% PEG 4000, 0.1 M Tris-HCl (pH
8.5), and 0.2 M magnesium chloride. (7) For the R203A/
V182A·BMP complex, the precipitant contained 1.0 M sodium
citrate and 0.1 M cacodylate (pH 6.5). (8) For the R203A/
T159V·BMP complex, the precipitant contained 0.8 M sodium
phosphate, 1.2 M potassium phosphate, and 0.1 M acetate (pH
4.5). (9) For the R203A/R160A·BMP complex, the precipitant
contained 3.5 M sodium formate (pH 7.0). (10) For the
T159V/V182A/Y206F·BMP complex, the precipitant con-
tained 60% tacsimate (pH 7.0).
Prior to data collection, the crystals of all 18 crystal forms
(Tables 1 and 2) were transferred to cryoprotectant solutions
composed of their mother liquids and 20% glycerol and flash-
cooled in a nitrogen stream. The X-ray diffraction data sets
(Tables 1 and 2) were collected at the NSLS X4A beamline
(Brookhaven National Laboratory, Upton, NY) on an ADSC
CCD detector and at the NSLS X29A beamline on the 315q
CCD detector. Diffraction intensities were integrated and
scaled with DENZO and SCALEPACK.27 The data collection
statistics are listed in Tables 1 and 2.
Structure Determination and Model Refinement. All
18 MtOMPDC structures (Tables 1 and 2) were determined by
molecular replacement with fully automated molecular
replacement pipeline BALBES,28 using only input diffraction
and sequence data. Partially refined structures of all 18
MtOMPDC crystal forms (Tables 1 and 2) were the outputs
from BALBES. Several subsequent iterative cycles of refinement
were performed for each crystal form, including: model
rebuilding with COOT,29 refinement with PHENIX,30 and
automatic model rebuilding with ARP.31
For all 18 BMP-liganded structures, (1) all loops are well-
defined, (2) the electron density for all BMP ligands is well-
ordered, (3) no non-glycine residue lies in disallowed regions of
the Ramachandran plots, (4) all crystallize as dimers, and (5)
the monomers within each dimer are connected by a
noncrystallographic two-fold axis.
Figure 4. Representative electron density map for the active site of the
Q185A mutant complexed with BMP and contoured at 1.5σ. The
figure was produced with PyMOL.41 The details of the interactions
between BMP and the active site are described in the text.
binding of the substrate, but is more completely realized in the
transition state.” For MtOMPDC, the 5′-phosphate group of
OMP “activates” decarboxylation 2.4 × 108-fold as measured by
comparing the values of kcat/Km for OMP and EO that lacks the
5′-phosphate group (Scheme 219,26,32) [2.9 × 106 and 12.4 ×
10−3 M−1 s−1, respectively (Table 3); the IBE calculated from
this ratio is 11.4 kcal/mol (Table 3)].
The challenge has been to provide a structure-based
understanding for how the IBE of a “remote” group of the
substrate is both generated and used to activate the enzyme.
Given the conformational change induced by ligand binding
(Figure 2), a plausible explanation is that in MtOMPDC the
IBE of the 5′-phosphate group not only increases the affinity of
the enzyme for the substrate [Ks (and Ks′) in the kinetic model
in Scheme 2 and Figure 3; green arrows in Figure 3] but also
increases the fraction of the enzyme that exists as Ec·S instead
of Eo·S (Kc ≫ 1 in contrast to Kc′ ≪ 1 for Ec and Eo; red and
blue arrows, respectively, in Figure 3) by allowing stabilizing
contacts between the “mobile” and “fixed” domains.
Model for Explaining the Role of the IBE of the 5′-
Phosphate Group in the Activation of MtOMPDC. We
previously proposed the kinetic model in Scheme 2 to
understand the relationships among Eo, Ec, S, and the values
of Km and kcat;19 the three-dimensional energy diagram for the
model is shown in Figure 3.
In the model, Eo predominates in the absence of substrate
(Kc′ ≪ 1); Ec predominates in its presence (Kc ≫ 1; as Ec·S).
The values of Kc′ and Kc are determined by the relative
stabilities of Eo and Ec in the absence and presence of substrate,
respectively. The dissociation constant for S, ([Eo][S])/[Ec·S],
Representative electron density for the Q185A mutant
liganded with BMP is shown in Figure 4.
Final crystallographic refinement statistics for all determined
MtOMPDC structures are listed in Tables 1 and 2.
a
is assumed to be the value of Km, 1/(KcKs) [=1/(Kc′Ks′)].
The value of the IBE for the 5′-phosphate group is obtained
from the increase in the value of kcat/Km associated with the 5′-
phosphate group occupying its binding site in Ec (from the ratio
of the values for OMP and EO). Because the carboxylate group
of the substrate (in either OMP or EO) is distal from the
binding site for the 5′-phosphate group (Figure 1), the value of
kcat (the energy difference between Ec·S and Ec·S⧧) can be
expected to be independent of the absence or presence of the
5′-phosphate group in the absence of “long-range” interactions
with the carboxylate group of the substrate and/or the anionic
RESULTS AND DISCUSSION
■
Intrinsic Binding Energy (IBE) of the 5′-Phosphate
Group. As described by Jencks,23 the IBE of a remote
substituent, e.g., the 5′-phosphate group of OMP, is used “to
pay for substrate destabilization through distortion, electrostatic
interactions, and desolvation, for bringing about the necessary
loss of entropy by freezing the substrates in the proper position
for reaction, and for binding to the transition state. The
maximum binding energy is then not realized directly in the
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Table 3. Kinetic Constants for OMP at pH 7.1 and 25 °C
kcat/Km
x-fold change in
x-fold change in
x-fold change in
cat/Km
ΔΔG⧧
b
MtOMPDC
wild type
kcat (s−1
)
Km (μM−1
1.8 0.5
)
(M−1 s−1
)
kcat
Km
k
(kcal/mol)
5.3 0.1
2.3 0.1
2.0 0.1
3.0 0.1
4.1 0.2
0.56 0.02
1.1 0.1
1.0 0.1
1.1 0.1
1.5 0.1
2.9 × 106
7.0 × 104
6.7 × 104
1.3 × 105
7.5 × 105
8.0 × 102
3.7 × 103
1.7 × 103
8.5 × 103
1.5 × 104
1.3 × 102
1.3 × 104
1.5 × 103
−
2
−
18
−
−
T159V
33
30
24
2
1
1
41
43
22
4
2.2
2.2
1.8
0.8
4.8
3.9
4.4
3.4
3.1
5.9
3.1
4.4
7.4
7.4
7.1
6.7
R160A
3
17
V182A
2
13
Y206F
5.5 1.0
700 90
300 50
600 60
130 10
100 15
1900 200
110 15
980 10
−
1
3
T159V/V182A
T159V/Y206F
R160A/V182A
R160A/Y206F
V182A/Y206F
10
5
390
170
330
72
3600
780
5
1700
5
340
4
56
190
T159V/V182A/Y206F 0.24 0.01
22
4
1100
61
2.2 × 104
220
Q185A
1.4 0.1
R203A
1.5 0.1
4
540
1900
a
Q185A/R203A
T159V/R203A
R160A/R203A
V182A/R203A
−
−
−
−
9.6
−
−
−
−
−
−
−
−
3.0 × 105
3.0 × 105
1.8 × 105
9.1 × 104
a
−
−
−
9.9
a
16
a
32
a
b
Velocity is directly proportional to substrate concentration. For kcat/Km.
allow the proposal that the hydrophobic cluster involving Val
182 in the active site loop and hydrogen bonds involving Thr
159, Arg 160, and Tyr 206 between the “mobile” and “fixed”
domains observed in Ec but not in Eo contribute to the stability
of Ec relative to Eo in the absence and presence of ligand [Kc′
and Kc, respectively (blue arrows in Figure 3)].
Scheme 3
In our initial studies of this model, we observed that Ala
substitutions for residues in the hydrophobic cluster (Val 182,
Ile 199, Val 201, and Ile 218) that are “remote” from the 5′-
phosphate group and its binding site increase the value of Km
but do not influence the value of kcat.19 We determined the
structures of the mutants in the presence of BMP and observed
that they can be superimposed on that of the wild type with the
exception of the side chains of the substituted residues. Thus,
the changes in the values of Km could be associated with an
incomplete “remote” hydrophobic cluster that reduces the
values of both Kc′ and Kc (blue arrows in Figure 3); the
unchanged values for kcat were explained by the unperturbed
interactions of the active site with the OMP substrate and
transition state in the Ec·S and Ec·S⧧ complexes (Figure 1), so
the energy difference between Ec·S and Ec·S⧧ is unchanged.
The Ala substitutions in the hydrophobic core do not alter
the values of the IBEs; i.e., the values of kcat/Km for OMP and
EO are equivalently reduced by the substitutions.19 Because the
values for EO measure the fraction of the unliganded enzyme
present as Ec (the value of Kc′), the equivalent reductions
demonstrate that the interaction of the 5′-phosphate group
with its binding site is not the sole determinant of the stability
of the Eo·S complex relative to Eo and S [Km = 1/(KcKs)] and
of Ec relative to Eo in the Ec·S and Eo·S complexes (Kc).
In the remainder of this work and in the context of this
model, we investigate the roles of the hydrogen bonding
interactions involving Thr 159, Arg 160, and Tyr 206 between
the “mobile” and “fixed” domains observed in Ec but not in Eo
as well as Gln 185 and Arg 203 that contribute to the binding
site for the 5′-phosphate group in the activation by the 5′-
phosphate group of OMP.
intermediate, i.e., the same value for OMP and EO if it were
possible to fully populate Ec·S when EO is the substrate. The
model also assumes that interaction of the 5′-phosphate group
is required to stabilize Ec·S relative to Eo·S (Kc vs Kc′ in the
unliganded enzyme), so the value of kcat/Km for EO is
determined by the fraction of the unliganded enzyme that exists
as Ec, i.e., from the value of Kc′.
The energy obtained from the 5′-phosphate group
interacting with its binding site (available to OMP but not
EO) can be used (1) to increase the stabilities of the Eo·S and
Ec·S complexes relative to Eo and S (green arrows in Figure 3;
“gripping” the substrate) and (2) to increase the stability of Ec·S
relative to Eo·S (red arrows in Figure 3; stabilizing the active
conformation). Both effects stabilize the Ec·S complex relative
to Eo and S, thereby decreasing the value of Km and increasing
the value of kcat/Km. Thus, the activation by the 5′-phosphate
group is caused by two independent effects that cannot be
deconvoluted: effects on Ks and/or Kc when OMP is the
substrate. If the value of kcat is independent of the interactions
of the 5′-phosphate group with its binding site [evaluated by
the loss of individual interactions by site-directed mutagenesis
(vide infra)], the activation by the 5′-phosphate group is the
result of (1) an increase in the affinity of the enzyme for the
OMP substrate (reflected in the value of Ks) and (2) an
increase in the fraction of the enzyme that exists as Ec (as Ec·S,
i.e., Kc vs Kc′).
The stability of Ec·S relative to Eo and S (measured by the
Km) is also influenced by residues “remote” from the binding
site for the 5′-phosphate group. The structures of the
unliganded and BMP-liganded structures of MtOMPDC
Identification of Interactions at the Domain Interface
That Stabilize Ec. MtOMPDC undergoes a conformational
change from Eo in the absence of a ligand to Ec in the presence
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of BMP. As discussed in the introductory section, this
conformational change involves closure of the active site loop
[Pro 180−Asp 188 (yellow backbone in Figure 2)] as well as
the movement of a “mobile” domain [β-strands 6−8 and 1 (red
α-helices and blue β-strands in polypeptide A in Figure 5)]
toward a “fixed” domain [β-strands 2−5 (magenta α-helices
and green β-strands)], with the “fixed” domains forming the
dimer interface.
Figure 6. Interactions formed by closure of the “mobile” and “fixed”
domains. (A) Hydrophobic cluster involving Val 182 at the N-
terminus of the active site loop. (B) Hydrogen bond between Tyr 206
and Lys 82* in the symmetry-related polypeptide in the dimer. (C)
Hydrogen bonds involving (1) the OH group of Thr 159 and the
backbone carbonyl oxygen of Ser 127 and (2) the guanidinium group
of Arg 160 and the backbone carbonyl oxygens of Met 126 and His
128.
Figure 5. Structure of the dimer of MtOMPDC liganded with BMP,
with the residues studied in this manuscript labeled. Ile 199, Val 201,
and Ile 218 that participate in the hydrophobic cluster near the N-
terminus of the active site loop (with Val 182) are also labeled.19
When the active site loop becomes ordered in Ec (yellow
backbone in Figures 2 and 5), the hydrophobic cluster
involving Val 182 in the active site loop is assembled [Val
182, Ile 199, Val 201, and Ile 21819 (cyan residues in Figure
5)], and hydrogen bonding contacts also “remote” from the
binding site for the 5′-phosphate group are established between
the “mobile” and “fixed” domains [Tyr 206 with Lys 82* in the
symmetry-related polypeptide in the dimer, Thr 159 with the
backbone carbonyl oxygen of Ser 127, and Arg 160 with the
backbone carbonyl oxygens of both Met 126 and His 128 (dark
cyan residues in Figure 5)]. Enlarged views of these interactions
are shown in Figure 6 (Gln 185 and Arg 203 that interact
directly with the 5′-phosphate group are colored brown). Our
hypothesis is that these “remote” interactions at the domain
interface also contribute to the stability of Ec relative to Eo as
we previously concluded for the hydrophobic cluster.19
Thr 159, Arg 160, and Tyr 206 in the “mobile” domain were
targeted, with the expectation that substitutions of these with
Ala (Thr 159 and Arg 160) or Phe (Tyr 206) would destabilize
Ec (as determined by the values of kcat/Km for EO). The
substitutions were not expected to alter the IBE, as previously
observed for the residues that participate in the hydrophobic
cluster at the base of the active site loop,19 because they are
“remote” from the binding site for the 5′-phosphate group.
Identification of Interactions That Generate the IBE of
the 5′-Phosphate Group. Each of the nonesterified oxygens
of the 5′-phosphate group participates in three hydrogen bonds
(a total of nine). As shown in Figure 7A, the 5′-phosphate
group of BMP directly contacts the carboxamide nitrogen of
the side chain of Gln 185 in the active site loop (via a single
hydrogen bond) and the guanidinium group of Arg 203 at the
end of the eighth β-strand in the “mobile” domain (via two
hydrogen bonds); it also contacts the backbone amide groups
of Gly 202 and Arg 203 (via a single hydrogen bond to each).
The importance of the interactions with the side chains of Gln
Figure 7. Hydrogen bonding interactions of the nonesterified oxygens
of the 5′-phosphate group of BMP. (A) Active site of MtOMPDC
(details provided in the text). (B) Active site of ScOMPDC (details
provided in the text). The water molecules that are involved in
hydrogen bonds to the 5′-phosphate groups are colored green.
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185 and Arg 203 in determining the activation by the 5′-
phosphate group can be quantitated by characterization of
mutants in which the side chains are “removed”, e.g., the
Q185A and R203A substitutions. However, the importance of
the interactions with the backbone amide groups cannot be
investigated by site-directed mutagenesis-based approaches.
In addition, the 5′-phosphate group is hydrogen-bonded to
four water molecules, with three participating in hydrogen
bonds to the backbone amide groups of Gly 181, Val 182, and
Gln 185 in the active site loop; the fourth water molecule is
hydrogen-bonded to both Tyr 206 in the “mobile” domain and
Asp 20 in the “fixed” domain. The importance of these
“indirect” contacts of the 5′-phosphate group with the protein
also is difficult to investigate using site-directed mutagenesis-
based approaches.
Our hypothesis is that all of the contacts made by the 5′-
phosphate group are used to generate the total interaction
energy that is partitioned between (1) “gripping” the substrate
[Ks in Scheme 2 and Figure 3 (green arrows)] and (2)
stabilizing both Ec·S and Ec·S⧧ (vide infra) relative to Eo·S [Kc
in Scheme 2 and Figure 3 (red arrows)]. When the active site
loop assumes its closed conformation when Gln 185 interacts
with the 5′-phosphate group and the hydrophobic cluster
involving Val 182 is formed, hydrogen bonding contacts
involving the loop’s backbone, including the carbonyl oxygen of
Pro 180, reposition the spatially proximal Thr 159 and Arg 160
in the “mobile” domain so that their side chains can form
hydrogen bonds across the domain interface to the backbone
carbonyl oxygens of Ser 127 as well as those of Met 126 and
His 128 that flank Ser 127 in the “fixed” domain. The
carboxamide oxygen of Gln 185 also forms a hydrogen bond
with the OH group of Ser 127 in the “fixed” domain. Thus, we
propose that IBE generated by the 5′-phosphate group is used,
in part, to allow assembly of the hydrophobic cluster at the base
of the active site loop as well as the interactions across the
domain interface, with these providing independent contribu-
tions to the stabilization of Ec·S and Ec·S⧧ relative to Eo·S
(Figures 5 and 6).
Gln 185 and Arg 203 were targeted for site-directed
mutagenesis, with the expectation that Ala substitutions
would destabilize Ec·S and Ec·S⧧ relative to Eo and S as judged
by a decrease in the IBE as the result of a reduction in the value
of kcat/Km for OMP but not for EO. Although the Q185A
mutation could be expected to decrease the value of Kc because
of the interaction of the carboxamide group with Ser 127 in the
“mobile” domain (Figure 1, as measured by the value of kcat/Km
for EO), the R203A mutation is not expected to alter the value
of Kc because it is “remote” from the domain interface.
Structures of Mutants of Residues That Stabilize Ec.
Five single mutants of residues that stabilize Ec (T159A, T159S,
T159V, R160A, and Y206F; V182A was previously described
and structurally characterized19) as well as five double mutants
and one triple mutant that combine four of these (T159V/
V182A, T159V/Y206F, R160A/V182A, R160A/Y206F,
V182A/Y206F, and T159V/V182A/Y206F) were constructed
and subjected to structural characterization in the presence of
BMP. In these structures, the catalytic triads, Asp 70, Lys 72,
and Asp 75* (from the symmetry-related polypeptide in the
dimer) as well as Ser 127 that hydrogen bonds to the
pyrimidine ring, superimpose well on the wild-type enzyme
(Figure 8). With the exception of the T159A substitution, the
only structural changes are those associated with the side chain
of the substituted residue; in the case of the T159A mutation,
Figure 8. Superposition of the BMP-liganded structures of wild-type
MtOMPDC and the K82A, T159V, R160A, Y206F, T159V/V182A,
T159V/Y206F, V182A/Y206F, and T159V/V182A/Y206F mutants of
residues that stabilize Ec: (A) polypeptide dimers and (B) structures of
the active sites.
the conformation of the loop flanking the substitution (Pro
157−Arg 163) is altered. Not surprisingly, the T159A mutant
displayed significantly compromised kinetic constants [the
value of kcat/Km is reduced 104-fold from that measured for the
wild type (data not shown)] compared to all of the other
mutants of residues that stabilize Ec. Our observation that the
values of kcat measured for the remaining mutants are
“unchanged” relative to that measured for the wild-type
enzyme is consistent with the observation that the structures
of the active sites are not perturbed by the substitutions.
Structures of Mutants of Residues That Generate the
IBE of the 5′-Phosphate Group. We constructed the Q185A
and R203 single substitutions as well as the Q185A/R203A
double substitution to remove the direct contacts between the
5′-phosphate group and the side chains of the protein. In the
structure of the Q185A/R203A double mutant, the active site
loop (Pro 180−Asp 188) is partially disordered in the A
polypeptide (Ala 184 and Ala 185, with increased B values for
the remaining “visible” loop residues) and assumes an altered
conformation (also with increased B values) in the B
polypeptide (Figure 9).
In the mutant proteins, the structures of the active sites are
not affected by the substitution(s); this is consistent with our
observation that the values of kcat for the Q185A and R203A
single mutants are “unchanged” from that measured for the
wild-type enzyme (vide infra). The value of kcat could not be
measured for the Q185A/R203A double mutant because the
value of Km is significantly elevated (Ks is significantly
decreased) by the loss of these contacts with the 5′-phosphate
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Figure 9. Superposition of the BMP-liganded structures of wild-type MtOMPDC and the Q185A, R203A, and Q185A/R203A mutants that generate
the IBE of the 5′-phosphate group: (A) polypeptide dimers, (B) active site of the wild type, (C) active site of the Q185A mutant, (D) active site of
the R203A mutant, and (E) active site of the Q185A/R203A mutant. The red spheres represent water molecules.
group. In the Q185A and Q185A/R203A mutants, two “new”
water molecules occupy the position of the carboxamide group
of Gln 185 in the wild-type enzyme: one is located in the same
position as the carboxamide amino group and is hydrogen-
bonded to one of the nonesterified phosphoryl oxygens, and
the second is located in the same position as the carboxamide
carbonyl oxygen and is hydrogen-bonded to both the first water
molecule and the OH group of Ser 127. This hydrogen-bonded
“chain” maintains an indirect interaction of the 5′-phosphate
group with the “fixed” domain and, therefore, is expected to
contribute to the stability of the domain interface, although the
carboxamide group is “missing”. Finally, in the R203A and
Q185A/R203A mutants, a “new” water molecule located in the
same position as the ε-nitrogen of the guanidinium group in the
wild-type protein is hydrogen-bonded to a second nonesterified
oxygen.
Structures of Double Mutants of Residues That
Stabilize Ec and Generate the IBE of the 5′-Phosphate
Group. The R203A substitution was combined separately with
the spatially remote T159V, R160A, and V182A substitutions.
As expected on the basis of the structures of the single
substitutions and the unchanged values for kcat (vide infra), the
structures of the active sites were unchanged in the mutants
(Figure 10). Also, as in the structure of R203A, in each
structure a “new” water molecule is located in the same position
as the ε-nitrogen of the guanidinium group in the wild-type
protein.
Kinetic Constants for Mutants of Residues That
Stabilize Ec. The values of kcat, Km, and kcat/Km using OMP
as the substrate were determined for the wild type and mutants
that contain “remote” substitutions for residues hypothesized to
stabilize Ec relative to Eo (Table 3). The values of kcat/Km for
EO and/or FEO, (kcat/Km)/KD for activation of decarbox-
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energy), providing support that these “remote” residues provide
“independent” contributions that stabilize Ec·S relative to Eo
and S.
The values of kcat/Km for EO for the single mutants are
decreased from that measured for the wild type; the calculated
values for the double and triple mutants are more significantly
reduced, with the changes again approximately multiplicative
relative to the values for the single mutants. The fact that the
order of magnitude decreases in the values of kcat/Km for OMP
and EO are equivalent reinforces the interpretation that the
targeted residues are important in determining the stability of
Ec; i.e., the substitutions give decreased values of Kc and Kc′
(blue arrows in Figure 3).
The values of (kcat/Km)/KD for activation of the decarbox-
ylation of EO by phosphite were determined for the single
mutants; these demonstrate that phosphite retains the ability to
activate the mutants. The fact that the values of kcat/Km for
OMP, kcat/Km for EO, and (kcat/Km)/KD for phosphite are
equivalently decreased is consistent with the interpretation that
the changes are determined by the values of Kc; i.e., the 5′-
phosphate group of OMP and phosphite both retain the ability
to increase the stability of Ec·S (and E·S⧧) relative to EoS (red
arrows in Figure 3)
As a result of the equivalent effects of the substitutions on the
values of kcat/Km for OMP and EO, the IBEs for the 5′-
phosphate group for the single and most of the double mutants
are unchanged from that measured for the wild-type enzyme;
the same observation was made for Ala substitutions for the
residues in the hydrophobic cluster.19 (For the T159V/V182A
and T159V/V182A/Y206F mutants, the values of the IBEs are
modestly decreased, likely the result of the same small
structural changes that may affect the values of kcat.) We
conclude that although the 5′-phosphate group provides the
same IBE, Ec·S and E·S⧧ are less stabilized for the mutants than
the wild-type enzyme because the interactions that stabilize the
closed conformation of the active site loop (Val 182) and
stabilize the domain interface (Thr 159, Arg 160, and Tyr 206)
have been disrupted.
Figure 10. Superposition of the BMP-liganded structures of wild-type
MtOMPDC and the T159V/R203A, R160A/R203A, and V182A/
R203A double mutants of residues that stabilize Ec and generate the
IBE of the 5′-phosphate group: (A) polypeptide dimers and (B)
structures of the active sites.
ylation of EO by phosphite (the ability of phosphite to mimic
the 5′-phosphate group) and the IBEs for the 5′-phosphate
group also were determined (Table 4).
Kinetic Constants for Mutants of Residues That
Generate the IBE of the 5′-Phosphate Group. Using
both OMP and EO, we measured the values of kinetic
constants for Q185A, R203A, and Q185A/R203A, the two
single mutants and one double mutant with substitutions for
the two residues with side chains that directly contact the 5′-
phosphate group of the substrate (Tables 3 and 4). With OMP,
the values of kcat and Km and, therefore, kcat/Km could be
measured for Q185A and R203A; however, only the value kcat/
Km could be measured for Q185A/R203A.
The values of kcat for the Q185A and R203A mutants are
“unchanged” from that measured for the wild-type enzyme
(Table 3), so the energy difference between the Ec·S and Ec·S⧧
complexes is not affected by the weakened ability of the
“remote” 5′-phosphate group to activate the enzyme. There-
fore, these substitutions alter the energies of both Ec·S and
Ec·S⧧ relative to Eo and S; i.e., the 5′-phosphate group does not
preferentially stabilize the transition state complex, Ec·S⧧, but
also equally stabilizes the Michaelis complex, Ec·S, because both
share the Ec conformation.
For the double and triple mutants, the more reactive FEO
was used to estimate the values of kcat/Km for EO.25,33,34 For
these mutants, the values of Km for OMP and, therefore, both
EO and FEO are significantly elevated because the fraction of
the enzyme that exists as Ec is reduced [Kc′ is reduced (blue
arrows in Figure 3)] because interactions that stabilize Ec are
absent. Because of the increased reactivity of FEO, lower
concentrations of the enzyme (and/or shorter reaction times)
can be used to measure the values of kcat/Km, thereby making
these measurements possible. The correction factor for the
double and triple mutants, 550, was obtained by averaging the
values of the ratios of kcat/Km for EO and FEO for wild-type
MtOMPDC and the single mutants (Table 4).
For the single mutants and most of the double mutants using
OMP as the substrate, the values of kcat are “unchanged”
relative to that measured for the wild-type enzyme; in contrast,
the values of Km are significantly increased. [The values of kcat
for OMP are (modestly) decreased for the T159V/V182A and
T159V/V182A/Y206F mutants, suggesting small changes in
structure and/or dynamics.] These observations are similar to
those we reported for Ala substitutions for the residues in the
hydrophobic cluster at the base of the active site loop that
includes Val 182.19 The increases in the values of Km for the
double and triple mutants are approximately multiplicative
relative to the values of the single mutants (additive in free
The values of kcat/Km for OMP are decreased, but the values
for EO are “unchanged” from that measured for the wild type.
The decreased value for OMP is explained by the weaker
affinity of the enzyme for the 5′-phosphate group, i.e., a
decrease in the value of Ks (green arrow in Figure 3). The fact
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Table 4. Kinetic Constants for EO and FEO and IBEs of the 5′-Phosphate Group of OMP at pH 7.1 and 25 °C
kcat/K for
m a
kcat/K for
mb
OMP
ΔΔG⧧
(kcal/mol) EO (M−1 s−1
kcat/Km for
ΔΔG⧧
FEO
(kcat/Km)/KD for
ΔΔG⧧
5′-phosphate
b
b
c
MtOMPDC
(M−1 s−1
)
)
(kcal/mol)
(M−1 s−1
)
FEO/EO EO·HPi (M−2 s−1
)
(kcal/mol) IBE (kcal/mol)
d
wild type
T159V
R160A
V182A
Y206F
2.9 × 106
7.0 × 104
6.7 × 104
1.3 × 105
7.5 × 105
8.0 × 102
−
2.2
12.4 × 10−3
0.4 × 10−3
0.2 × 10−3
1.4 × 10−3
6.0 × 10−3
6.2 × 10−5
−
4.8
390
210
2300
35
-
11.4
11.2
11.6
10.8
11.0
9.6
2.0
2.4
1.3
0.4
3.1
0.083
0.21
0.63
2.2
2.4
2.1
1.6
1.2
2.2
1.8
0.8
4.8
1100
470
56
150
280
370
e
e
e
e
e
e
T159V/
V182A
0.034
T159V/
Y206F
3.7 × 103
1.7 × 103
8.5 × 103
1.5 × 104
1.3 × 102
3.9
4.4
3.4
3.1
5.9
6.3 × 10−5
4.6 × 10−5
5.8 × 10−5
5.5 × 10−4
3.1 × 10−5
3.1
3.3
3.1
1.8
3.5
0.035
0.025
0.032
0.3
10.5
10.3
11.1
10.1
9.0
R160A/
V182A
R160A/
Y206F
V182A/
Y206F
T159V/
V182A/
Y206F
0.017
Q185A
R203A
1.3 × 104
1.5 × 103
9.6
3.1
4.4
7.4
2 × 10−3
5.5 × 10−3
3.2 × 10−3
1.0
0.4
0.8
0.92
4.7
470
860
24
2.7
4.0
9.2
7.4
4.7
2.4
Q185A/
R203A
T159V/
R203A
9.9
16
32
7.4
7.1
6.7
0.2 × 10−3
0.2 × 10−3
2.4
2.4
1.3
6.4
6.6
5.9
R160A/
R203A
V182A/
R203A
1.3 × 10−3
a
b
c
d
From Table 3. The estimated error is 10%. Calculated from the ratio of the values of kcat/Km for OMP and EO. A lower estimate because the
value of kcat/Km for OMP is partially diffusion-controlled.24 Calculated from the value of kcat/Km for FEO by dividing the value by 550 (the average
e
ratio of the values of kcat/Km for EO and FEO for the wild type and those mutants for which the value for EO could be reliably measured).
that the values for EO are unchanged provides evidence that
Gln 185 and Arg 203 are not involved in determining the
relative energies of Eo and Ec. Thus, we hypothesize that the
Saccharomyces cerevisiae (ScOMPDC).35 In ScOMPDC, the 5′-
phosphate group interacts with the side chains of three
residues, Gln 215 and Arg 235, structural homologues of Gln
185 and Arg 203, respectively, in MtOMPDC, and Tyr 217 in
the longer (19-residue) active site loop (Figure 7B). In
ScOMPDC, the 5′-phosphate group is hydrogen-bonded to the
backbone amide groups of Gly 234 and Arg 235, structural
homologues of Gly 202 and Arg 203, respectively, in
MtOMPDC. Also, in ScOMPDC, the 5′-phosphate group is
hydrogen-bonded to three (not four) water molecules, two
hydrogen-bonded to the backbone of the active site loop and
the third hydrogen-bonded to Asp 37, the structural homologue
of Asp 20 in MtOMPDC.
Kinetic analyses for single and multiple substitutions of the
residues that contact the 5′-phosphate group (Q215A, Y217F,
and R235A) revealed progressive decreases in the values of the
IBE, with the values of kcat/Km for OMP being only 12-fold
greater than that for EO for the Q215A/Y217F/R235A triple
mutant. An explanation for why the direct contacts with the
side chains apparently are “fully” responsible for generating the
IBE in ScOMPDC but not in MtOMPDC is not obvious.
However, the active site loops differ in both length and
conformation; also, structures are not available for the mutants
of ScOMPDC, so unlike MtOMPDC, the effects of the
substitutions on the structure of the protein and active site
cannot be assessed.
c
IBE allows the conformational reorganization of the active site
loop that allows formation of the interdomain contacts
involving Thr 159, Arg 160, and Tyr 206 as well as assembly
of the hydrophobic cluster involving Val 182 in the active site
loop, with these determining the stability of Ec relative to Eo.
The value of the IBE of the 5′-phosphate group for the wild-
type enzyme is 11.4 kcal/mol (Table 4). For the Q185A and
R203A mutants, the values are decreased by 2.1 and 4.0 kcal/
mol, respectively; for Q185A/R203A, the value is decreased by
6.7 kcal/mol (approximately the sum of those for the single
mutants). However, despite the complete absence of the direct
contacts of the 5′-phosphate group with side chains, the
Q185A/R203A mutant is activated 2900-fold by the 5′-
phosphate group (the IBE is 4.7 kcal/mol). The 5′-phosphate
group retains its interactions with the backbone amide groups
of Gly 202 and Arg 203 as well as several ordered water
molecules, including one that also forms hydrogen bonds with
Asp 20 in the “fixed” domain (Figure 7A).
The values of (kcat/Km)/KD for activation of the decarbox-
ylation of EO by phosphite were also determined for the single
mutants. The values are decreased significantly, as expected
because the values of Ks for phosphite binding should be
decreased by the absence of these direct interactions.
The fact that the side chain contacts with the 5′-phosphate
group are not entirely responsible for generating the IBE of the
5′-phosphate group contrasts with results we reported for
similar mutagenesis-based experiments with the OMPDC from
Kinetic Constants for Double Mutants of Residues
That Stabilize Ec and Generate the IBE of the 5′-
Phosphate Group. To obtain evidence that the “remote”
interactions that stabilize Ec·S and Ec·S⧧ relative to Eo·S and
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those that generate the IBE of the 5′-phosphate group by direct
contacts are independent, we constructed and characterized the
R203A/T159V, R203A/R160A, and R203A/V182A double
mutants. We chose the R203A instead of the Q185A
substitution because Gln 185 is hydrogen-bonded not only to
the 5′-phosphate group but also to the OH group of Ser 127 in
the “mobile” domain; i.e., Gln 185 could contribute to both
stabilization of Ec (and, therefore, the value of Kc′) and
generation of the IBE. In fact, the Q185A substitution does
olecular and requires no cofactors, the wild-type enzyme and
mutants can be structurally characterized, a sensitive direct
spectrophotometric assay is available, and substrate analogues
(EO and FEO) can be used to “isolate” the role of the
activating 5′-phosphate group. Prior structural characterization
established that substrate binding in MtOMPDC is accom-
panied by a conformational change in which a catalytically
active, closed conformation (Ec) that is unfavorable in the
absence of the substrate predominates when the substrate is
bound. We now have obtained persuasive kinetic and structural
evidence that the IBE of the 5′-phosphate group provides the
energy for a local reorganization of the structure of its binding
site in the Eo·S complex (in particular, the ordering of the active
site loop that includes Gln 185) that then allows the formation
of additional “remote” interactions at the interface between the
“mobile” and “fixed” domains that provide the energy to
stabilize Ec in both the Ec·S and Ec·S⧧ complexes. As a result of
this essential conformational change in which Ec, the
catalytically active conformation, is unstable relative to Eo, the
unliganded conformation, product release is more favorable
than if the total energy obtained from substrate binding were
used “simply” to bind the substrate. We expect that this strategy
will be applicable to understanding the structural basis for the
activation of other enzymes that undergo an essential
conformational change from an open, inactive conformation
(Eo) to a closed, active conformation (Ec) as the result of
substrate binding.
b
cause a 6-fold decrease in the value of kcat/Km for EO. The
kinetic constants for these mutants also are included in Tables
3 and 4.
The values of kcat and Km for OMP could not be measured
for these mutants: because all of the single mutants affect the
stabilities of Ec·S relative to Eo and S, the values of Km were
expected to be and are, in fact, too large to be measured (Table
3). The values of kcat/Km for EO for the double mutants are
essentially identical to those measured for the T159V, R160A,
and V182A single mutants that alter the stability of Ec·S relative
to Eo; i.e., the value of kcat/Km for EO for the R203A mutant is
“unchanged” (only 2-fold decreased) from that measured for
the wild-type enzyme (Table 4). Thus, the effects of the
substitutions are additive with respect to stabilization of Ec in
both Ec·S and Ec·S⧧. Also, the values for the IBE for the 5′-
phosphate group are essentially the same as that measured for
the R203A mutant; i.e., the effects of the substitutions on the
IBE also are additive (Table 4).
Independent Structural Strategies To Allow and
Accomplish the Stabilization of Ec. Taken together, our
structural and kinetic observations provide persuasive evidence
that the model presented in Scheme 2 and Figure 3 provides a
structure-based understanding of how the IBE of the 5′-
phosphate group is both generated and used to activate the
enzyme. The direct interactions of the 5′-phosphate group with
the side chains of Gln 185 in the active site loop and Arg 203
[as well as the backbone amide groups of Gly 202 and Arg 203
and water molecules that interact with the active site loop
(green arrows in Figure 3)] organize the active site loop (red
arrows) so that the hydrophobic cluster at the base of the active
site loop and the “remote” interdomain interactions can be
formed to stabilize Ec·S and Ec·S⧧ relative to Eo·S (blue arrows).
This “cascade” of structurally expanding interactions,
triggered by the binding of OMP to Eo and likely orchestrated
by changes in the structure of the active site loop directed by
the “remote” 5′-phosphate group, results in the remarkable 2.4
× 108-fold rate enhancement as the stabilizing and/or
destabilizing interactions are enforced in Ec·S and Ec·S⧧.
Further studies of interactions of the 5′-phosphate group
with its binding site, including the importance of the water-
mediated contacts with the active site loop, are in progress.
Our studies using both the intact OMP substrate and EO or
FEO that lacks the 5′-phosphate group19,25,26,32 provide a
kinetics- and structure-based approach for understanding the
role of the substrate and the IBE of its pieces in allowing the
conformational changes that are required for efficient enzymatic
catalysis. The dynamic characteristics of conformational
changes that accompany catalysis have been studied in other
enzymes, including adenylate kinase36,37 and dihydrofolate
reductase.38−40 The principles that emerge from the kinetics-
based approaches described here and in previous studies of
triosephosphate isomerase22 as well as dynamics-based
approaches that employ time-resolved NMR spectroscopy,
isotope exchange, and simulations together (1) provide both a
description and an understanding of Nature’s structural
strategies for achieving large rate accelerations and (2) inform
the (re)design of enzymic catalysts for novel reactions by
revealing the requirements for binding of the substrate to Eo,
utilization of energy provided by binding interactions with
“remote” portions of the substrate to, in part, effect a necessary
conformational transition from Eo·S to Ec·S that allows catalysis
via Ec·S⧧, and, finally, a favorable conformational relaxation to
Eo to permit product dissociation.
ASSOCIATED CONTENT
Accession Codes
■
CONCLUSIONS
■
The importance of the IBEs of “remote” substituents in
achieving the transition state stabilization that are responsible
for the impressive rate enhancements of enzyme-catalyzed
reactions has long been recognized.23 However, a unifying
structure-based description and understanding for how the IBE
of remote substituents is generated and used to effect transition
state stabilization has been elusive. Like triosephosphate
isomerase,22 MtOMPDC provides a useful system for exploring
and defining how the IBE of the “remote” phosphate group of
the substrate is generated and used to contribute to its
impressive 1017-fold rate acceleration: the reaction is unim-
The X-ray coordinates and structure factors for the following
structures have been deposited in the Protein Data Bank: the
K82A mutant of MtOMPDC in the presence of BMP (entry
3RLU), the T159A mutant of MtOMPDC in the presence of
BMP (entry 3P5Y), the T159S mutant of MtOMPDC in the
presence of BMP (entry 3P5Z), the T159V mutant of
MtOMPDC in the presence of BMP (entry 3P60), the
R160A mutant of MtOMPDC in the presence of BMP (entry
3P61), the Q185A mutant of MtOMPDC in the presence of
BMP (entry 3V1P), the R203A mutant of MtOMPDC in the
presence of BMP (entry 3LI0), the Y206F mutant of
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MtOMPDC in the presence of BMP (entry 3RLV), the
T159V/V182A mutant of MtOMPDC in the presence of BMP
(entry 3QEZ), the T159V/R203A mutant of MtOMPDC in
the presence of BMP (entry 4FXR), the T159V/Y206F mutant
of MtOMPDC in the presence of BMP (entry 3QF0), the
R160A/V182A mutant of MtOMPDC in the presence of BMP
(entry 3QMR), the R160A/R203A mutant of MtOMPDC in
the presence of BMP (entry 4GC4), the R160A/Y206F mutant
of MtOMPDC in the presence of BMP (entry 3SJ3), the
V182A/R203A mutant of MtOMPDC in the presence of BMP
(entry 4FX6), the V182A/Y206F mutant of MtOMPDC in the
presence of BMP (entry 3QMT), the Q185A/R203A mutant of
MtOMPDC in the presence of BMP (entry 4FX8), and the
T159V/V182A/Y206F mutant of MtOMPDC in the presence
of BMP (entry 3QMS).
substrate binding favors the transition of Eo·S to Ec·S is
conserved, although the details are not conserved.
b
The only possible pathway from Eo to Ec·S is via [Eo·S]
because the closed active site loop in Ec prevents binding of S
to Ec. Also, Km = 1/(KcKs) because Km is defined as a
dissociation complex but Ks is defined as an association
constant.
c
For Q185A, the value of kcat/Km for EO is reduced 6-fold,
perhaps reflecting the hydrogen bonding of the carboxamide
group to the OH group of Ser 127 in the “mobile” domain.
However, as described previously, the structure of the Q185A
mutant reveals that the carboxamide group is replaced by two
“new” water molecules that form a hydrogen bond chain
between the 5′-phosphate group and Ser 127.
REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Institute for Genomic Biology, University of Illinois, 1206 W.
Gregory Dr., Urbana, IL 61801. Phone: (217) 244-7414. Fax:
(217) 333-0508. E-mail: j-gerlt@uiuc.edu.
■
■
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Funding
†This research was supported by National Institutes of Health
(NIH) Grants GM039754 (to J.P.R.) and GM065155 (to
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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).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to the staff of NSLS beamlines X4A and X29A
for their help with the collection of diffraction data.
■
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ABBREVIATIONS
■
BMP, l-(5-phospho-β-ribofuranosyl)barbituric acid; FOMP, 5-
fluoroorotidine 5′-monophosphate; EO, 1-(β-D -
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ADDITIONAL NOTES
All OMPDCs that have been structurally characterized in the
■
a
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