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

摘要

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 °C and pD 7.0 – 9.3. Kinetic analysis of deuterium exchange gives pKa ≤ 22 for carbon deprotonation of enzyme-bound >UMP, which is at least 10 units lower than that for deprotonation of an analog 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-fold enzymatic rate acceleration.