Investigation of Substrate Specificity in Phosphate Binding Barrels and Mechanistic Studies of Orotidine Monophosphate Decarboxylase
Chan, Kui K.
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https://hdl.handle.net/2142/84865
Description
Title
Investigation of Substrate Specificity in Phosphate Binding Barrels and Mechanistic Studies of Orotidine Monophosphate Decarboxylase
Author(s)
Chan, Kui K.
Issue Date
2006
Doctoral Committee Chair(s)
Gerlt, John A.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
"Orotidine 5'-monophosphate decarboxylase from Methanobacterium thermoautotrophicum (MtOMPDC) catalyzes the decarboxylation of orotidine 5'-monophosphate (OMP) and uridine 5'-monophosphate (UMP) with an enormous rate acceleration, 1.8 x 1016 (kcat = 5 s-1, 25°C; knon = 2.8 x 1016 s-1), and proficiency, 7.1 x 1021 M-1 (kcat /KM = 2.0 x 106 M-1 s -1, 25°C), without employing metal ions or other cofactors. Despite attracting considerable attention amongst mechanistic enzymologists, the mechanism is still unresolved. I now report substantial evidence that decarboxylation facilitated by substrate destabilization resolves the formation of a carbanion intermediate in a two-step mechanism: (1) the product isotope effect (PIE) is unity; (2) the proton at C6 of [6- 1H]-uridine 5'-monophosphate (h-UMP) exchanges to give [62H]-uridine 5'-monophosphate (d-UMP) in solvent deuterium and; (3) differing effects of k cat and kex are observed by substitutions for Asp 70 in MtOMPDC. The rate of exchange of H6 of the UMP product estimates the active site stabilize the vinyl carbanion intermediate by at least 14 kcal/mol and accounts for a substantial part of the rate acceleration (≥ 1010). The kinetic study of D70G mutant in MtOMPDC of the Asp 70 substitution study suggests that the decarboxylation step by substrate destabilization could contribute approximately 5 kcal/mol. As a result, I have successfully accounted for the majority of the rate enhancement in the reaction catalyzed by OMPDC. I have also begun to investigate the structural basis of the two-step mechanism catalyzed by MtOMPDC. I report that a cooperativity between Ser 127 and Gln 185 of MtOMPDC, provides a ""switch"" for coupling substrate binding to catalysis. I also report that numerous structures suggest that conformational changes/dynamics couple to decarboxylation during catalysis. Even though a two-step mechanism with a carbanion intermediate is secured, further research to understand the structural basis for stabilization of the carbanion intermediate and the structural basis involved in conformational changes/dynamics will be needed."
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