Transimination Reaction at the Active Site of Aspartate Aminotransferase: A Proton Hopping Mechanism through Pyridoxal 5′-Phosphate

Abstract

The transimination reaction involves conversion of an internal aldimine involving pyridoxal 5′-phosphate (PLP) and an enzyme to an external aldimine involving PLP and a substrate amino acid and it constitutes an essential step in many biological processes catalyzed by PLP-dependent enzymes. We have investigated the free energy landscape and mechanistic pathways of the transimination process at the active site of aspartate aminotransferase by means of hybrid quantum–classical molecular dynamics simulations combined with various enhanced sampling techniques. It is found that, after a geminal diamine is formed in the first step of the process, the reaction proceeds through a path where a proton from the amine nitrogen of the substrate amino acid is transferred first to the phenolic oxygen of the PLP ring, and from there, it is transferred to the imine nitrogen of the active site lysine in the next step of the reaction. Both of these proton transfer events are found to be assisted by relative rotation of the PLP ring which brings the phenolic oxygen of PLP closer to the amine and imine nitrogens of the substrate and lysine, respectively. The transfer of the proton from the phenolic oxygen of PLP to the active site lysine residue is found to be the rate-determining step with an effective barrier of only 4 kcal/mol. Neither any direct proton transfer from lysine to the substrate nor any indirect proton transfer involving any active site residue or water is found.