Protein Structure and Catalytic Mechanism of Amino Acid Racemase

氨基酸消旋酶的蛋白质结构及催化机制

基本信息

批准号：
63580152
负责人：
TANIZAWA Katsuyuki
金额：
$ 1.09万
依托单位：
Osaka University (1989)Kyoto University (1988)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

1. The following results have been obtained by the research on glutamate racemase. (1) Glutamate racemase has been purified from cell extracts of a lactic acid bacterium, Pediococcus pentosaceus, and its enzymological properties and reaction mechanism have been analyzed. The enzyme catalyzed an internal proton transfer under single turnover conditions with a deuterium-labeled substrate or deuterium oxide as the solvent. (2) The gene coding for glutamate racemase has been cloned into Escherichia coli. An efficient method was established for purification of the enzyme from the clone cell extracts. (3) A convenient procedure has been developed for the enantioselective synthesis of various D-amino acids by a multi-enzyme system including glutamate racemase and D-amino acid transaminase.2. The following results have been obtained by the research on alanine racemase. (1) The structural gene coding for thermostable alanine racemase of a mesothermophilic bacterium, Bacillus stearothermophilus, … More has been cloned and expressed in E. coli. The enzyme was purified to homogeneity from cell extracts of E. coli carrying a plasmid designated pICR4. The alanine racemase gene sequenced was found to contain an open reading frame of 1158 nucleotides. The molecular weight of the enzyme subunit was estimated to be 43,341. (2) The alpha-helical and beta-structure contents were calculated to be about 34 and 26%, respectively, from CD data. CD measurements of the denaturation process of enzyme by guanidine hydrochloride showed the presence of a stable intermediate during the denaturation. It has been suggested that the enzyme subunit is composed of two structurally dissimilar domains connected by a short polypeptide. (3) A synthetic DNA fragment has been inserted into the alanine racemase gene at the position corresponding to the hinge region. The gene was expressed in E. coli to produce an active enzyme with a molecular structure similar to the wild-type enzyme. (4) Single crystals of alanine racemase were prepared by the polyethylene glycol vapor diffusion method. X-ray crystallographic analysis is currently in progress. Less

1.通过对谷氨酸消旋酶的研究，获得了以下结果： (1)从乳酸菌戊糖片球菌细胞提取物中纯化出谷氨酸消旋酶，并对其酶学性质和催化反应机理进行了分析。使用氘标记底物或氧化氘作为溶剂在单周转条件下进行内部质子转移 (2) 编码的基因。谷氨酸消旋酶已被克隆到大肠杆菌中。建立了从克隆细胞提取物中纯化该酶的方法。(3)开发了一种通过多酶高效系统对映选择性合成各种D-氨基酸的简便方法。包括谷氨酸消旋酶和D-氨基酸转氨酶。2．对丙氨酸消旋酶的研究取得了以下成果：(1)耐热性编码基因的结构。嗜热脂肪芽孢杆菌的丙氨酸消旋酶已在大肠杆菌中克隆并表达，该酶从携带 pICR4 质粒的大肠杆菌细胞提取物中纯化至同质。经测序，发现含有丙氨酸消旋酶基因。 1158个核苷酸的开放阅读框估计酶亚基的分子量为43,341. 根据盐酸胍酶变性过程的 CD 数据，计算出 α 螺旋和 β 结构含量分别约为 34% 和 26%。有人认为该酶亚基由两个结构不同的结构域组成，通过短多肽连接 (3) 合成的 DNA 片段已插入丙氨酸消旋酶中。 (4)通过聚乙二醇蒸气制备丙氨酸消旋酶单晶。 X射线晶体分析目前正在进行中。