Mechanisms of Decarboxylation: Internal Return, Water Addition, and Their Isotope Effects

2-(2-mandelyl)thiamin (MTh), the adduct of benzoylformate and thiamin, is an accurate model of 2-(2-mandelyl)thiamin diphosphate, the initial covalent intermediate in the decarboxylation of benzoylformate by benzoylformate decarboxylase (BFDC). The first order rate constant for spontaneous decarboxylation of MTh is about 106 times smaller than the enzymic rate (kcat) for the BFDC reaction. Based on the similarities of MTh and the corresponding enzymic intermediate, as well as the inherent nature of the intermediate, it is not obvious why the enzyme-catalyzed reaction is so much faster. However, earlier studies showed that the decarboxylation of MTh is catalyzed by protonated pyridines and this was proposed to occur through a preassociation mechanism. If this explanation is correct, then the observed 12C/13C kinetic isotope effect (CKIE) will increase in the presence of the catalyst as a more favorable forward commitment is made possible. This provides a specific model for the enzyme-catalyzed process. We developed a technique using headspace analysis and compound specific isotope analysis (CSIA) to determine the CKIE for the decarboxylation of MTh in the presence and absence of pyridinium. We found that the CKIE increases in the presence of the catalyst, as predicted for the preassociation mechanism. In a related study, we investigated the kinetics of decarboxylation of pyrrole-2-carboxylic acid, which was known to be subject to acid catalysis in highly acidic solutions. In the expected mechanism, protonation of the pyrrole ring at C2 destroys the aromaticity of the ring. C-C bond cleavage in the process of decarboxylation will re-establish the aromatic pyrrole. However, the overall reaction rate would not increase as it is counteracted by a larger concentration of the undissociated carboxyl group compared to carboxylate ion necessary for decarboxylation. Since the reaction occurs readily, there must be an alternative pathway for the acid-catalyzed reaction. This can be achieved in an associative mechanism that is initiated by addition of water to the carboxyl group of the carboxyl-protonated reactant. C-C bond cleavage results in formation of pyrrole and protonated carbonic acid, a species that has been recognized as a viable intermediate in related processes. Protonated carbonic acid is spontaneously converted to H3O+ and carbon dioxide. The associative mechanism is consistent with solvent-deuterium kinetic isotope effects and 12C/13C kinetic isotope effects.Ph