Transient adducts of CO2 with nucleophiles enhance rates of enzymic decarboxylation.

Benzoylformate decarboxylase (BFDC) forms an adduct of thiamin diphosphate (ThDP), and benzoylformate, following the mechanism proposed by Breslow for related enzymes. Structural studies by Hasson and coworkers revealed inactivation of the enzyme by reaction with benzoylphosphonate, a substrate analogue that produces a phosphate ester of the active site serine (Ser26). This suggests that the side chain of Ser26 can trap CO2 as a transient hemicarbonate that prevents the reverse reaction that captures CO2, accelerating the forward reaction toward the ultimate release of benzaldehyde. Through enzyme mutagenesis, Ser26 variants containing other nucleophilic side chains were prepared and analyzed for their reaction with the inhibitor benzoylphosphonate to determine the extent of inactivation. The results indicate the native enzyme and its variants have nearly equal rates of inactivation by the phosphate-based inhibitor despite drastic differences in the enzyme rate constant. This implies a dissociative PO3- transfer mechanism that leads to inactivation at similar rates through formation of a phosphate of each nucleophilic variant. We propose the variation in rate constant between the nucleophilic side chains is the result of decomposition of the hemicarbonate ester formed in decarboxylation. This suggests that the enzyme's rate-limiting process is not the formation of CO2 but rather involves its eventual release from the reaction site. Overall, the results imply that blocking reversion of CO2 is a key factor that establishes the enzyme's proficiency.