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PDBsum entry 3gdk
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References listed in PDB file
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Key reference
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Title
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Mechanism of the orotidine 5'-Monophosphate decarboxylase-Catalyzed reaction: evidence for substrate destabilization.
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Authors
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K.K.Chan,
B.M.Wood,
A.A.Fedorov,
E.V.Fedorov,
H.J.Imker,
T.L.Amyes,
J.P.Richard,
S.C.Almo,
J.A.Gerlt.
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Ref.
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Biochemistry, 2009,
48,
5518-5531.
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PubMed id
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Abstract
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The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC)
involves a stabilized anionic intermediate, although the structural basis for
the rate acceleration (k(cat)/k(non), 7.1 x 10(16)) and proficiency
[(k(cat)/K(M))/k(non), 4.8 x 10(22) M(-1)] is uncertain. That the OMPDCs from
Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae
(ScOMPDC) catalyze the exchange of H6 of the UMP product with solvent deuterium
allows an estimate of a lower limit on the rate acceleration associated with
stabilization of the intermediate and its flanking transition states
(>or=10(10)). The origin of the "missing" contribution, <or=10(7) (
approximately 10(17) total - >or=10(10)), is of interest. Based on structures
of liganded complexes, unfavorable electrostatic interactions between the
substrate carboxylate group and a proximal Asp (Asp 70 in MtOMPDC and Asp 91 in
ScOMPDC) have been proposed to contribute to the catalytic efficiency [Wu, N.,
Mo, Y., Gao, J., and Pai, E. F. (2000) Proc. Natl. Acad. Sci. U.S.A. 97,
2017-2022]. We investigated that hypothesis by structural and functional
characterization of the D70N and D70G mutants of MtOMPDC and the D91N mutant of
ScOMPDC. The substitutions for Asp 70 in MtOMPDC significantly decrease the
value of k(cat) for decarboxylation of FOMP (a more reactive substrate analogue)
but have little effect on the value of k(ex) for exchange of H6 of FUMP with
solvent deuterium; the structures of wild-type MtOMPDC and its mutants are
superimposable when complexed with 6-azaUMP. In contrast, the D91N mutant of
ScOMPDC does not catalyze exchange of H6 of FUMP; the structures of wild-type
ScOMPDC and its D91N mutant are not superimposable when complexed with 6-azaUMP,
with differences in both the conformation of the active site loop and the
orientation of the ligand vis a vis the active site residues. We propose that
the differential effects of substitutions for Asp 70 of MtOMPDC on
decarboxylation and exchange provide additional evidence for a carbanionic
intermediate as well as the involvement of Asp 70 in substrate destabilization.
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