Designing composite biocatalysts by analysis of the proton-transfer process in pyridoxal enzymes

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By analyzing the proton-transfer process of pyridoxal enzymes, we elucidated the refined catalytic reaction mechanisms of them, thereby presenting the molecular understanding of the "induced fit" and "multisubstrate recognition". In aspartate aminotransferase (AAT), the conformational hange to the closed form induces an electrostatic repulsion between the main chain carbonyl group of Gly38 and the Schiff base, thereby raising the ree energy level of the Michaelis complex and increasing the k_<cat> value. Kinetic and structural analysis on the reaction of AAT with C5 substrates in comparison with C4 substrates revealed that in the case of AAT-C5 the Michaelis complex takes the open conformation and the external aldimine complex takes the closed conformation, while in AAT-C4, both complexes take the closed conformation. In the closed conformation, There is a hydrophobic interaction between the residues that is located at the active site entrance and covers the active site after the substrate binding. In the Michaelis complex with C5, the loss of this hydrophobic interaction increases the free energy level of the Michaelis complex, again contributing to the increase in the k_cat Value for C5 substrates. In this way, we revealed the systematic interactions between the parts--catalytic part, recognition part, regulation part, and so on--of composite biocatalysts.