We report that orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes exchange of the C-6 proton of uridine 5'-monophosphate (UMP) for deuterium from solvent in D2O at 25 degrees C and pD 7.0-9.3. Kinetic analysis of deuterium exchange gives pK(a)<= 22 for carbon deprotonation of enzyme-bound UMP, which is atleast 10 units lower than that for deprotonation of an analogue of UMP in water. The observation of enzyme-catalyzed deuterium exchange via a stabilized carbanion provides convincing evidence for the decarboxylation of orotidine 5'-monophosphate (OMP) 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 OMP, and that this transition state stabilization constitutes a large fraction, but probably not all, of the enormous 10(17)-fold enzymatic rate acceleration.