Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Effect of Solvent Viscosity on Kinetic Constants

摘要

Orotidine 50n-monophosphate decarboxylase (OMPDC) is an exceptionally proficient catalyst: thenrate acceleration (kcat/knon) is 7.1 u00021016n, and the proficiency [(kcat/KM)/knon] is 4.8 u00021022nM-1n. The structuralnbasis for the large rate acceleration and proficiency is unknown, although themechanismhas been establishednto involve a stabilized carbanion intermediate. To provide reaction coordinate context for interpretation ofnthe values of kcat, kcat/KM, and kinetic isotope effects, we investigated the effect of solvent viscosity on kcat andnkcat/KM for the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomycesncerevisiae (ScOMPDC). ForMtOMPDC, we used not only the natural OMP substrate but also a catalyticallynimpaired mutant (D70N) and a more reactive substrate (FOMP); for ScOMPDC, we used OMP and FOMP.nWith MtOMPDC and OMP, kcat is independent of solvent viscosity, indicating that decarboxylation is fullynrate-determining; kcat/KM displays a fractional dependence of solvent viscosity, suggesting that both substratenbinding and decarboxylation determine this kinetic constant. For ScOMPDC with OMP, we observed thatnboth kcat and kcat/KM are fractionally dependent on solvent viscosity, suggesting that the rates of substratenbinding, decarboxylation, and product dissociation are similar. Consistentwith these interpretations, for bothnenzymes with FOMP, the increases in the values of kcat and kcat/KM are much less than expected based on thenability of the 5-fluoro substituent to stabilize the anionic intermediate; i.e., substrate binding and productndissociation mask the kinetic effects of stabilization of the intermediate by the substituent