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

通过对吡哆醛类酶的质子转移过程的分析，阐明了它们的精细催化反应机理，从而展示了对“诱导FIT”和“多底物识别”的分子理解。在天冬氨酸氨基转移酶(AAT)中，由于构象改变为闭合形式，导致Gly38主链上的羰基与Schiff碱之间发生静电斥力，从而提高了Michaelis络合物的REE能级，增加了k&lt；CAT&gt；值。AAT与C5底物和C4底物反应的动力学和结构分析表明，在AAT-C5中，Michaelis络合物为开放构象，外部乙二胺络合物为闭合构象，而在AAT-C4中，两种络合物均为闭合构象。在封闭构象中，位于活性中心入口的残基与底物结合后覆盖活性中心的残基之间存在疏水相互作用。在与C5的Michaelis络合物中，这种疏水相互作用的丧失增加了Michaelis络合物的自由能级，再次导致了C5底物的kcat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.