Product deuterium isotope effect for orotidine 5′-monophosphate decarboxylase: Evidence for the existence of a short-lived carbanion intermediate

A product isotope effect (PIE) of 1.0 was obtained from the ratio of the yields of [6-¹H]-uridine 5′-monophosphate (50%) and [6-²H]-uridine 5′-monophosphate (50%) from the decarboxylation of orotidine 5′-monophosphate (OMP) in 50/50 (v/v) H₂O/D₂O catalyzed by orotidine 5′-monophosphate decarboxylase. This unitary product isotope effect eliminates a proposed mechanism for enzyme-catalyzed decarboxylation in which proton transfer from Lys-93 to C-6 of OMP provides electrophilic push to the loss of CO₂ in a concerted reaction. The complete lack of selectivity for the reaction of solvent H and D that is implied by the value of PIE = 1.0 may be enforced by restricted motion of the NL₃⁺ group of the side-chain of Lys-93 that has been proposed to protonate a vinyl carbanion intermediate.