From the Department of Biochemistry and Biophysics,
University of North Carolina, Chapel Hill, North Carolina
27599-7260 and ¶ Molecular Sciences, GlaxoWellcome, Research
Triangle Park, North Carolina 27709
Received for publication, December 19, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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The crystal structure of yeast
orotidine-5'-phosphate decarboxylase in complex with the
postulated transition state analog, 6-hydroxyuridine-5'-phosphate,
reveals contacts between this inhibitor and a novel quartet of charged
residues (Lys-59, Asp-91, Lys-93, and Asp-96) within the active site.
The structure also suggests a possible interaction between
O2 of the 6-hydroxyuridine-5'-phosphate pyrimidine
ring and Gln-215. Here we report the results of mutagenesis of each of
the charged active site residues and Gln-215. The activities of the
Q215A and wild-type enzymes were equal indicating that any interactions
between this residue and the pyrimidine ring are dispensable for
efficient decarboxylation. For the D91A and K93A mutant enzymes,
activity was reduced by more than 5 orders of magnitude and substrate
binding could not be detected by isothermal calorimetry. For the D96A
mutant enzyme, kcat was reduced by more than 5 orders of magnitude, and isothermal calorimetry indicated an 11-fold
decrease in the affinity of this enzyme for the substrate in the
ground state. For the K59A enzyme, kcat was
reduced by a factor of 130, and Km had increased by
a factor of 900. These results indicate that the integrity of the
network of charged residues is essential for transition state stabilization.
Orotidine-5'-phosphate decarboxylase
(ODCase;1 EC 4.1.1.23)
produces a large rate enhancement, accelerating the rate of spontaneous decarboxylation of orotic acid derivatives by more than 17 orders of
magnitude (see Ref. 1 and Fig. 1). The remarkable catalytic power of
this enzyme is entirely dependent on non-covalent binding forces and
does not involve metals or other cofactors (2, 3). In an attempt to
gain insight into its mechanism of action, the crystal structures of
ODCase complexed with three different ligands were recently determined
(4-7). In each of these structures, a unique arrangement of
alternating, charged residues composed of Lys-Asp-Lys-Asp is positioned
near the bound pyrimidine ring of the ligand. These four residues,
Lys-59, Asp-91, Lys-93, and Asp-96 of the yeast enzyme (see Fig. 2),
are completely conserved in all known ODCase sequences (8). In the
present work, we sought to determine their roles in catalysis.
The spontaneous decarboxylation of orotidine-5'-phosphate is
very slow indeed (knon = 2.8 × 10 Based on computer simulations, it has been proposed (5, 6)
that Asp-91 (yeast numbering system) may assist decarboxylation by a
mechanism involving electrostatic destabilization of the substrate in
the ground state. However, the likelihood of that mechanism of
destabilization has been questioned (10). Lys-93 has been shown to be
important for enzyme activity (9), possibly by stabilizing the
carbanion produced by CO2 elimination and furnishing the proton that appears at C-6 of the product UMP (Fig.
1). The second acidic member of the
charged quartet, Asp-96, is contributed by the opposite subunit of the
ODCase dimer (4-7) and is drawn into the active site by ligand
binding. The final member of the quartet, Lys-59, appears to contact
the 3'-hydroxyl group of the bound substrate (5-7).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
16 sec
1). According to the theory of
absolute reaction rates, a large value of
kcat/Km (for ODCase = 6.3 × 107 M
1
sec
1) requires that the altered substrate be very tightly
bound in the transition state Ktx < 10
23 M (1). Moreover, a large value of
kcat (for ODCase = 44 s
1)
requires that the substrate be much less tightly bound in the ground
state (1), as was recently established (Km = Ks = 7 × 10
7 M)
(11, 13). From a structural standpoint, it would be desirable to
understand how these very different affinities are achieved.
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Fig. 1.
The reaction catalyzed by
orotidine-5'-phosphate decarboxylase.
To assess the contribution of each of these four residues to
ground state and transition state stabilization, we have replaced each
one with alanine. We also substituted alanine for Gln-215, a residue
that appears to be within hydrogen-bonding distance (2.6 Å) of the
C-2 oxygen of the BMP pyrimidine ring, possibly aiding
delocalization of negative charge in the transition state for
decarboxylation (4). Here we show that removal of any member of the
charged network drastically reduces activity. In contrast, mutagenesis
of Gln-215 reveals that this residue contributes very little to
transition state stabilization.
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EXPERIMENTAL PROCEDURES |
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Mutant recombinant ura3 genes encoding
K59A, D91A, K93A, D96A, and Q215A enzymes were generated by
site-directed mutagenesis using Quick-Change reagents (Stratagene,
Inc.) and mutagenic oligonucleotide pairs (Oligos, Etc.). Wild-type and
mutant enzymes were expressed and purified from Escherichia
coli SS6130 (cytR, cdd) as previously described (11). Analysis of each mutant protein, either intact or as
protease-generated peptides, by electrospray mass spectrometry demonstrated the correct alanyl substitution at positions 59, 91, 93, 96, and 215. Enzymatic decarboxylation of OMP was measured in
MOPS buffer (2.0 × 10
2 M, pH 7.2) by
observing the decrease in absorbance at either 285 or 295 nm where
M =
1743 and
819 cm
1, respectively.
An alternative assay (12), monitoring the evolution of
14CO2 from radiolabeled OMP, was used to
estimate values of Km for wild-type and Q215A
enzymes. Concentrations of wild-type and mutant enzymes were estimated
from absorbance readings at 280 nm, using a molar extinction
coefficient of 28,830 cm
1. Ligand binding affinities were
determined by comparing rates of decarboxylation in the presence and
absence of varying concentrations of each competitive inhibitor.
Isothermal titration calorimetry was performed on a Microcal, Inc.
MSC calorimeter equilibrated at 25 °C with a final enzyme
concentration of 10
4 M. Circular dichroism
spectra were obtained at 25 °C using an Applied Photophysics
* 180 spectrophotometer following dilution of each enzyme
into potassium phosphate buffer (10
2 M, pH
7.2).
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RESULTS AND DISCUSSION |
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The crystal structure of yeast ODCase complexed with BMP
suggested the possibility of a hydrogen bond between Gln-215 and O2 of the pyrimidine ring (see Ref. 4 and Fig.
2). However, the data in
Tables I and
II show that replacement of Gln-215 with alanine scarcely affects either catalysis or ligand binding. This result is surprising considering the closeness of the Gln-215 side
chain to O2 of the pyrimidine ring and indicates that any interactions between this residue and the pyrimidine ring are dispensable for efficient decarboxylation. Aside from contacts with the
6-substituent, the only remaining contact between the enzyme and
the pyrimidine ring is formed between O-4 of the pyrimidine ring and
the peptide bond amide group of Ser-154 (4).
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Table III compares the activity of
wild-type yeast ODCase with the activities of the K59A, D91A, K93A, and
D96A mutant enzymes. In these assays, which contained substrate at a
concentration 350-fold greater than the Km value for
wild-type ODCase (2.5 × 104 M),
no detectable activity was observed for the D91A, K93A, or D96A mutant
enzymes, even at an enzyme concentration up to 2.0 × 10
4 M in subunits. These experiments
indicated that the activity of each mutant enzyme had decreased by more
than 5 orders of magnitude compared with the wild-type enzyme (Table
III). The loss of activity resulting from mutagenesis of Asp-91,
Lys-93, and Asp-96 does not appear to be a consequence of gross changes
in enzyme structure, as indicated by comparison of the CD spectra of
mutant and wild-type enzymes.
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In view of the negligible levels of enzyme activity observed for these
mutant enzymes, it was of interest to determine whether they were
capable of binding OMP. For the D91A and K93A mutant proteins, OMP
binding could not be detected by isothermal titration calorimetry. In
the case of the D96A protein, isothermal titration calorimetry yielded
a value of 8.0 × 106 M for the binding
affinity of OMP (Table I). This value is similar to the
Km value of the wild-type enzyme for
2'-deoxyorotidine 5'-phosphate determined in a separate
study.2 Earlier work
has shown that, for the wild-type enzyme, Km represents the dissociation constant of the enzyme-substrate complex (11, 13). The ability of the D96A protein to bind ligand, in contrast
to the results obtained for the D91A and K93A proteins, is of interest
in view of the fact that Asp-96 is contributed to the active site by
the opposite subunit of dimeric ODCase upon ligand binding. Based on
the activity limit and substrate binding affinity of the D96A protein,
the apparent kcat/Km value for this mutant enzyme was reduced by more than 6 orders of magnitude. The absence of detectable substrate binding by the D91A and K93A mutant
enzymes precludes the assessment of the effects of these substitutions
upon the stability of the ES complex. However, the lack of activity of
the D91A, K93A, and D96A mutant enzymes indicates that each of these
charged residues play a critical role in transition state stabilization.
The crystal structures of all ODCase-ligand complexes suggest the
presence of a hydrogen bond between the -amino group of Lys-59 and
the 3'-OH group of the ligand (4-7). Whereas removal of the
side-chains of Asp-91, Lys-93, and Asp-96 completely destroyed activity, the enzyme retained measurable activity after mutagenesis of
Lys-59. Table I shows that the kcat value for
the K59A mutant enzyme was reduced 100-fold whereas
Km was elevated 1000-fold compared with these
parameters for the wild-type enzyme.
Table IV reveals a striking disparity
between the magnitude of the effect of the Lys-59 Ala substitution
on the binding affinities of the substrate in the ground state and the
altered substrate in the transition state. As the reaction progresses from the ground state ES complex to the transition state, the effect of
the K59A mutation increases from 910-fold to 1.2 × 105-fold. Upon formation of product UMP, the magnitude of
this effect collapses to less than 2-fold. These findings are
consistent with the view that the active site of this enzyme is
organized to maximize the influence of multiple binding interactions in
the transition state, while minimizing such effects in the ground state
and product complexes. The differential effect of the Lys-59
Ala
substitution upon ligand binding is comparable with, but somewhat
exceeds, the effect of removing the 2'-OH group of the ligand,
consistent with the view that interactions between enzyme and ribosyl
OH groups are important for transition state stabilization.
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To the extent that enzymatic decarboxylation depends on ground state destabilization, one would expect that any enzyme mutation that reduces this destabilization would tend to both reduce kcat and increase the affinity of the enzyme for the substrate in the ground state. In fact, both the K59A and D96A mutant enzymes show reduced values of kcat and reduced affinities for the substrate in the ground state. Similarly, both the D91A and K93A mutants were inactive, and isothermal titration calorimetry experiments ruled out the possibility of an increased affinity for the substrate.
To enhance the rate of any reaction over the rate of reaction that is observed in water, an enzyme binds the altered substrate in the transition state more tightly than it binds the substrate in the ground state ES complex (14). This difference in affinities may be achieved, at least in part, by the introduction of a local strain in the ES complex that is relieved as the ES complex proceeds toward the transition state.3
In the case of ODCase, it has been suggested that repulsive
interactions between Asp-91 and the carboxylate group of OMP may produce ground state destabilization by electrostatic interactions, which are relieved in the transition state as CO2 is
eliminated (5). If this enzyme acted by such a mechanism, with ground state repulsion present in the ES complex with UMP, one might expect
significantly tighter binding of product OMP in which such repulsion
would be absent. In fact, the affinity of the enzyme for product UMP
(Ki = 2.0 × 107 M)
is greatly exceeded by its affinity for the substrate OMP, whose
Km value (7 × 10
7 M)
has been shown to be a true dissociation constant (11, 13). These
affinities, combined with the present effects of active site
modification, seem to indicate unequivocally that the active site binds
the substrate strongly in the ground state and that their mutual
affinity increases greatly as the enzyme-substrate complex progresses
toward the transition state.
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ACKNOWLEDGEMENTS |
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We thank W. Burkhart, M. Moyer, and K. Blackburn for electrospray mass spectrometric and amino acid sequence analysis of wild-type and mutant enzymes.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM-18324 and National Institutes of Health Training Grant GM-08570.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence may be addressed. E-mail: water@med.unc.edu.
To whom correspondence may be addressed. E-mail:
sas44336@glaxowellcome.com.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011429200
2 Miller, B. G., Butterfoss, G. L., Short, S. A., and Wolfenden, R. (2001) Biochemistry, in press.
3 It is worth noting that the introduction of ground state destabilization elevates kcat and Km to the same extent, without enhancing kcat/Km. Most enzymes operate with the substrate at sub-saturating concentrations (15), and the introduction of ground state destabilization does not enhance the rate of reaction under these conditions.
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ABBREVIATIONS |
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The abbreviations used are: ODCase, orotidine-5'-phosphate decarboxylase; BMP, 6-hydroxyuridine-5'-phosphate; OMP, orotidine 5'-phosphate; MOPS, 4-morpholinepropanesulfonic acid; ES, enzyme-substrate.
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