Investigation of the carboligase activity of thiamine diphosphate-dependent enzymes using kinetic modeling and NMR spectroscopy

The benzoin condensation reaction catalyzed by the thiamine diphosphate (ThDP)-dependent enzymes benzaldehyde lyase (BAL) and benzoylformate decarboxylase variant His281Ala (BFDH281A) was studied via initial rate measurements, progress curve analysis and NMR-based analysis of reaction intermediates. Using a mechanistic kinetic model, the kinetic parameters and microscopic rate constants were determined, thus identifying the rate limiting steps of the reaction. In BAL, overall reaction is rate-limited by product release, whereas in BFDH281A substrate binding is the slowest step of catalysis. These results were further confirmed by analysis of covalent reaction intermediates using NMR spectroscopy after acid quench isolation.