Formation and stability of a vinyl carbanion at the active site of orotidine 5'-monophosphate decarboxylase: pKa of the C-6 proton of enzyme-bound UMP.

We report that orotidine 5′-monophosphate decarboxylase (OMPDC) catalyzes exchange of the C-6 proton of uridine 5′-monophosphate () for deuterium from solvent in DO at 25 °C and pD 7.0 – 9.3. Kinetic analysis of deuterium exchange gives p ≤ 22 for carbon deprotonation of enzyme-bound , which is at least 10 units lower than that for deprotonation of an analog of in water. The observation of enzyme-catalyzed deuterium exchange via a carbanion provides convincing evidence for the decarboxylation of orotidine 5′-monophosphate () by OMPDC to give the same carbanion intermediate. The data show that yeast OMPDC stabilizes the bound vinyl carbanion by at least 14 kcal/mol. We conclude that OMPDC also provides substantial stabilization of the late carbanion-like transition state for the decarboxylation of , and that this transition state stabilization constitutes a large fraction, but probably not all, of the enormous 10-fold enzymatic rate acceleration.