Protein Engineering of Aspartate Aminotransferase

天冬氨酸氨基转移酶（AspAT）是由维生素B_6衍生物5 '-磷酸吡哆醛（Pyridoxal 5'-phosphate）作为辅助因子，催化L-天冬氨酸和2-酮戊二酸之间的可逆氨基转移。建立了研究AspAT催化机理的体系。测定了大肠杆菌AspAT基因的核苷酸序列。通过定点突变替换活性位点中的氨基酸残基。野生型和突变型AspAT在E.大肠杆菌菌株（TY 103）缺乏AspAT基因。测定了野生型和突变型AspATs的三维结构，停流法测定了辅酶吸附量的变化，阐明了AspATs的催化过程AspAT的半转氨反应包括两个快速平衡的底物结合步骤和一个分子内速率决定步骤，在速率决定步骤中，Lys 258、Tyr 70、Try 225和Asp 222，在底物结合过程中，观察到四种有趣的现象。(1)Arg对于识别底物的羧基是必不可少的。赖氨酸不能代替精氨酸。(2)当Arg 292被瓦尔或Leu取代时，AspAT的底物特异性由酸性变为中性。(3)AspAT的活性位点是疏水性的。(4)AspAT有两个口袋用于容纳底物的侧链，一个用于酸性底物（Asp，Glu），另一个用于疏水底物。

Aspartate aminotransferase (AspAT) has the essential cofactor, pyridoxal 5'-phosphate (a vitamin B_6 derivative) and catalyzes the reversible amino group transfer between L-aspartate ad 2-oxoglutarate. The system for studying the catalytic mechanism of AspAT was constructed. The nucleotide sequence of the gene for Escherichia coli AspAT was determined. The amino acid residues in the active site were replaced by site-directed mutagenesis. The wild-type and mutant AspATs were overproduced in the E. coli strain (TY103) lacking the gene for AspAT. The three-dimensional structures of the wild-type and mutant AspATs were determined.A stopped-flow method monitoring the absorption change of the coenzyme clarified that the catalytic process (half-transamination reaction) of AspAT consists of the two substrate-binding steps in rapid equilibrium and an intramolecular rate-determining step.In the rate-determining step, Lys258, Tyr70, Try225, and Asp222, which exist around the bound coenzyme, played important roles.In the substrate binding, four interesting phenomena were observed.(1) Arg is essential for recognizing the carboxyl group of the substrate. Lys cannot substitute for Arg.(2) When Arg292 was replaced by Val or Leu, the substrate specificity of AspAT was changed from acidic to neutral substrates.(3) The active site of AspAT is hydrophobic.(4) AspAT has two pockets for accomomdating the side chain of a substrate, one for acidic substrate (Asp, Glu), and the other for hydrophobic substrate.