Orotidine Monophosphate Decarboxylase: Study of the Mechanism of Stabilization of the Reaction Intermediate.

摘要

Orotidine 5'-monophosphate decarboxylase (OMPDC) is unique in its exceptional proficiency and mechanism of decarboxylation. There is substantial experimental evidence that the OMPDC-catalyzed decarboxylation of OMP proceeds through a two-step mechanism involving the formation of a carbanion intermediate. The work presented in this dissertation is a continuation of the enormous effort towards understanding the mechanism by which OMPDC enhances the rate of the reaction by 1017-fold in comparison to the nonenzymatic reaction. In particular, the focus of the dissertation is on elucidating the detailed mechanism by which the enzyme provides such an effective stabilization of the carbanion reaction intermediate, which in turn effects this extraordinary rate enhancement. The experimental approach involved the synthesis of several specifically designed phosphodianion truncated substrate/product analogs, 1-(beta-D-erythrofuranosyl)-5-fluorouracil (FEU), 1-(beta-D-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5'-deoxy-5-fluoroorotidine (5'-dFO), and determination of the kinetic parameters for their reactions with wild type and/or R235A mutant enzymes. The findings from these experiments determined the following: (1) the degree of stabilization provided by the remote substrate phosphodianion group on the transition state for the OMPDC-catalyzed deuterium exchange reaction of FUMP (the reaction in the reverse direction), (2) the stabilizing effect of the 5-F substituent on the developing negative charge at the neighboring C-6 atom for the enzyme-catalyzed decarboxylation of OMP (the reaction in the forward direction), (3) the effect of the 4'-CH 3 substituent on the stability of the transition state for the enzymatic decarboxylation of FEO, and (4) the role of the interactions between the guanidinium cationic side chain of Arg235 and the substrate phosphodianion in stabilizing the vinyl carbanion intermediate for the OMPDC-catalyzed deuterium exchange reaction of FUMP. The experimental data from these experiments were compared to data from previous studies and used to construct a correct kinetic model which best describes the mechanism of action of OMPDC.