Protein Structure and Catalytic Mechanism of Amino Acid Racemase

1.通过对谷氨酸消旋酶的研究，获得了以下结果。(1)从戊糖片球菌（Pediococcus pentosaceus）细胞提取物中分离纯化了谷氨酸消旋酶，并对其酶学性质和反应机理进行了研究。该酶催化的内部质子转移在单周转条件下与氘标记的底物或氘氧化物作为溶剂。(2)编码谷氨酸消旋酶的基因已被克隆到大肠杆菌中。建立了一种从克隆细胞提取物中纯化该酶的有效方法。(3)本研究建立了一种利用谷氨酸消旋酶和D-氨基酸转氨酶的多酶体系对映体选择性合成多种D-氨基酸的简便方法.通过对丙氨酸消旋酶的研究，获得了以下结果。(1)编码嗜热脂肪芽孢杆菌耐热丙氨酸消旋酶的结构基因， ...更多信息 在E.杆菌从大肠杆菌的细胞提取物中纯化该酶至均一。携带命名为pICR 4的质粒的大肠杆菌。丙氨酸消旋酶基因含有一个1158个核苷酸的开放阅读框。酶亚基的分子量估计为43，341。(2)从CD数据计算的α-螺旋和β-结构含量分别为约34%和26%。用圆二色谱法研究了盐酸胍对酶的变性过程，结果表明在变性过程中存在一个稳定的中间体。有人认为，该酶亚基是由两个结构不同的结构域由一个短的多肽连接。(3)合成的DNA片段已插入丙氨酸消旋酶基因中对应于铰链区的位置。该基因在E.大肠杆菌，以产生具有与野生型酶相似的分子结构的活性酶。(4)用聚乙二醇气相扩散法制备了丙氨酸消旋酶单晶。X射线晶体学分析目前正在进行中。少

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