Role of the Carboxylate in Enzyme-Catalyzed Decarboxylation of Orotidine 5'-Monophosphate: Transition State Stabilization Dominates Over Ground State Destabilization.

Kinetic parameters (s) and / (M s) are reported for exchange for deuterium in DO of the C-6 hydrogen of 5-fluororotidine 5'-monophosphate () catalyzed by the Q215A, Y217F, and Q215A/Y217F variants of yeast orotidine 5'-monophosphate decarboxylase (OMPDC) at pD 8.1, and by the Q215A variant at pD 7.1-9.3. The pD rate profiles for wildtype OMPDC and the Q215A variant are identical, except for a 2.5 log unit downward displacement in the profile for the Q215A variant. The Q215A, Y217F and Q215A/Y217F substitutions cause 1.3-2.0 kcal/mol larger increases in the activation barrier for wildtype OMPDC-catalyzed deuterium exchange compared with decarboxylation, because of the stronger apparent side chain interaction with the transition state for the deuterium exchange reaction. The stabilization of the transition state for the OMPDC-catalyzed deuterium exchange reaction of is ca. 19 kcal/mol smaller than the transition state for decarboxylation of , and ca. 8 kcal/mol smaller than for OMPDC-catalyzed deprotonation of to form the vinyl carbanion intermediate common to OMPDC-catalyzed reactions and . We propose that OMPDC shows similar stabilizing interactions with the common portions of decarboxylation and deprotonation transition states that lead to formation of this vinyl carbanion intermediate, and that there is a large ca. (19-8) = 11 kcal/mol stabilization of the former transition state from interactions with the nascent CO of product. The effects of Q215A and Y217F substitutions on / for decarboxylation of are expressed mainly as an increase in for the reactions catalyzed by the variant enzymes, while the effects on / for deuterium exchange are expressed mainly as an increase in . This shows that the Q215 and Y217 side chains stabilize the Michaelis complex to for the decarboxylation reaction, compared with the complex to for the deuterium exchange reaction. These results provide strong support for the conclusion that interactions which stabilize the transition state for OMPDC-catalyzed decarboxylation at a nonpolar enzyme active site dominate over interactions that destabilize the ground-state Michaelis complex.