权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Protein Engineering of Aspartate Aminotransferase

天冬氨酸转氨酶的蛋白质工程

基本信息

批准号：
01570144
负责人：
KURAMITSU Seiki
金额：
$ 1.41万
依托单位：
Osaka Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

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.

天冬氨酸氨基转移酶(ASPAT)具有重要的辅因子5‘-磷酸吡哆醛(维生素B_6衍生物)，催化L-天冬氨酸和2-氧戊二酸之间的可逆氨基转移。构建了研究ASPAT催化机理的体系。测定了该基因的核苷酸序列。活性部位的氨基酸残基被定点突变取代。野生型和突变型Aspat在缺乏Aspat基因的大肠杆菌菌株(TY103)中大量生产。测定了野生型和突变型Aspat的三维结构。停流法监测辅酶的吸收变化，发现Aspat的催化过程(半转氨化反应)由两个快速平衡的底物结合步骤和分子内速率决定步骤组成。在速率决定步骤中，存在于结合辅酶周围的Lys258、Tyr70、Try225和Asp222起重要作用。在底物结合中，观察到四个有趣的现象。(1)Arg是识别底物羧基的关键。(2)当Arg292被Val或Leu取代时，Aspat的底物专一性从酸性底物变为中性底物。(3)Aspat的活性中心是疏水的。(4)Aspat有两个口袋，一个容纳底物的侧链，一个容纳酸性底物(Asp，Glu)，另一个容纳疏水底物。