Exploring the factors influencing the ketoenamine-enolimine tautomeric equilibrium of pyridoxal 5′-phosphate in branched-chain aminotransferases

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is a critical coenzyme for various enzymes. It generates a Schiff base with the substrate and exhibits ketoenamine and enolimine tautomeric forms due to intramolecular proton transfer. This study aims to ascertain the predominant tautomeric form of the PLP Schiff base in MtIlvE and analyze its influencing factors. Molecular dynamics simulations indicate that the ketoenamine tautomer has higher binding free energy than the enolimine tautomer. Density functional theory calculations suggest that, despite their ability to interconvert at a relatively low energy barrier, the ketoenamine tautomer is thermodynamically more stable. Factors affecting the keto-enol tautomeric equilibrium were investigated by constructing various QM-cluster models. Our results demonstrate that both the protonation of the pyridine nitrogen and the presence of Tyr209, which stabilizes the O3 anion, shift the tautomeric equilibrium toward the ketoenamine configuration. These findings provide a theoretical basis for investigating enzyme catalytic mechanisms.