Unraveling the biocatalytic mechanism of dipeptide formation using QM/MM and stopped-flow kinetic experiments

The mechanism of dipeptide formation catalyzed by β-aminopeptidase (DmpA) was investigated using a combination of computational and experimental approaches in this study. Using L-carnosine as a dipeptide model, real-time kinetic insights into the reaction mechanism and intermediates were obtained for the first time by stopped-flow UV-Vis spectroscopy combined with Singular Value Decomposition (SVD) analysis. Quantum Mechanics/Molecular Mechanics (QM/MM) calculations were used to determine the energy values of the intermediate and transition states, revealing the formation of three intermediates and providing critical insights into the formation of CN bonds during aminolysis, which was identified as the rate-limiting step. The QM/MM-calculated free energy of activation (ΔG^‡ = 39.20 kcal mol^-1) was consistent with the lowest rate constant (k₃ = 3.580 × 10^-2 s^-1) obtained from SVD analysis. The stopped-flow freeze-quenching technique was used to capture intermediate species, and their structures were elucidated using nuclear magnetic resonance studies. These findings provide a detailed mechanistic understanding of DmpA-catalyzed dipeptide formation.