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

通过分析吡哆醛酶的质子转移过程，阐明了吡哆醛酶的精细催化反应机理，从而提出了对“诱导契合”和“多底物识别”的分子理解。天冬氨酸氨基转移酶（AAT）中Gly 38的构象转变为闭合型时，Gly 38的主链羰基与Schiff碱之间产生静电排斥，从而使米氏复合物的能级升高，k值增大<cat>。对AAT与C5底物反应的动力学和结构分析表明，在AAT-C5底物中，米氏络合物为开放构象，醛亚胺络合物为封闭构象;在AAT-C4底物中，米氏络合物和醛亚胺络合物均为封闭构象。在闭合构象中，位于活性位点入口的残基之间存在疏水相互作用，并在底物结合后覆盖活性位点。在具有C5的米氏络合物中，这种疏水相互作用的丧失增加了米氏络合物的自由能水平，再次有助于C5底物的k_cat值的增加。通过这种方式，我们揭示了复合生物催化剂的催化部分、识别部分、调节部分等部分之间的系统相互作用。

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.