Orotidine 5′-monophosphate decarboxylase (OMPDC) is an exceptionally proficient catalyst: the rate acceleration (kcat/knon) is 7.1 × 1016, and the proficiency [(kcat/K M)/knon] is 4.8 × 1022M-1. The structural basis for the large rate acceleration and proficiency is unknown, although the mechanism has been established to involve a stabilized carbanion intermediate. To provide reaction coordinate context for interpretation of the values of kcat, kcat/KM, and kinetic isotope effects, we investigated the effect of solvent viscosity on kcat and kcat/KM for the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC). For MtOMPDC, we used not only the natural OMP substrate but also a catalytically impaired mutant (D70N) and a more reactive substrate (FOMP); for ScOMPDC, we used OMP and FOMP. With MtOMPDC and OMP, kcat is independent of solvent viscosity, indicating that decarboxylation is fully rate-determining; kcat/KM displays a fractional dependence of solvent viscosity, suggesting that both substrate binding and decarboxylation determine this kinetic constant. For ScOMPDC with OMP, we observed that both k cat and kcat/KM are fractionally dependent on solvent viscosity, suggesting that the rates of substrate binding, decarboxylation, and product dissociation are similar. Consistent with these interpretations, for both enzymes with FOMP, the increases in the values of kcat and kcat/KM are much less than expected based on the ability of the 5-fluoro substituent to stabilize the anionic intermediate; i.e., substrate binding and product dissociation mask the kinetic effects of stabilization of the intermediate by the substituent.
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