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Comparative Studies on Structure and Function of Pyridoxal Dependent and Indepandent Amino Acid Racemases

吡哆醛依赖性和独立氨基酸消旋酶结构和功能的比较研究

基本信息

批准号：
04680188
负责人：
ESAKI Nobuyoshi
金额：
$ 1.28万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04680188/
关键词：
Alanine Racemase Amino Acid Rasemase with Low Substrate Apecificity Hydroxyproline Epimerase Glutamate Racemase Aspartate Racemase Pyridoxal Phosphate

项目摘要

Two distinct types of amino acid racemases are known : one requires pyridoxal phosphate as a coenzyme and is represented by alanine racemase, the other requires neither cofactors nor coenzymes and is represented by glutamate racemase. It is interesting and important to compare catalytic mechanisms of these two types of enzymes, both of which catalyze racemization of alph-amino acids. We aimed to study the structures and functions of both types of enzymes, thereby to clarify mechanisms of the amino acid racemization. As to pyridoxal phosphate dependent amino acid racemases, we have chosen thermostable alanine racemase from Bacillus stearothermophilus. We have constructed a mutant enzyme of it in which Lys39 binding the coenzyme pyridoxal phosphate is replaced by alanlne, purified it, and characterized its enzymological properties. In particular, we have studied spectrophotometric properties of the coenzyme complexes and reaction with beta-chloroalanine. Pyridoxal-dependent amino acid racemase with low substrate specificity from Pseudomonas putida has been studied as well. We have cloned its structural gene, purified the enzyme from the clone cells, characterized the enzyme, determined the stereochemistry of hydrogen transfer in the abortive transamination, and studied the reaction mechanism. On the other hand, as to pyridoxal phosphate-independent racemases, glutamate racemase and aspartate racemase, we studied the role of cysteine residues of both enzymes by site-directed mutagenesis and chemical modification. Glutamate racemase loses the ability to transfer alph-proton, when either Cys73 or Cys184 is replaced by Ala or modified. On the other hand, apsartate racemase is completely inactivated by modification or replacement of only one Cys73 per dimeric enzyme.Therefore, we have proposed that asparate racemase probably has a composit active site.

已知两种不同类型的氨基酸外消旋酶：一种需要磷酸吡哆醛作为辅酶，以丙氨酸外消旋酶为代表；另一种既不需要辅助因子也不需要辅酶，以谷氨酸外消旋酶为代表。比较这两种酶的催化机制是很有趣和重要的，这两种酶都能催化α -氨基酸的外消旋化。我们旨在研究这两种酶的结构和功能，从而阐明氨基酸外消旋化的机制。对于磷酸吡哆醛依赖的氨基酸外消旋酶，我们选择了嗜热脂肪芽孢杆菌中的耐热丙氨酸外消旋酶。我们构建了一种以丙烯取代Lys39结合磷酸吡哆醛辅酶的突变酶，对其进行了纯化，并对其酶学性质进行了表征。特别地，我们研究了辅酶配合物的分光光度性质和与-氯丙氨酸的反应。对恶臭假单胞菌低底物特异性的吡哆醛依赖性氨基酸消旋酶也进行了研究。我们克隆了它的结构基因，从克隆细胞中纯化了酶，对酶进行了表征，测定了流产转氨化过程中氢转移的立体化学性质，并研究了反应机理。另一方面，对于吡哆醛磷酸非依赖性外消旋酶、谷氨酸外消旋酶和天冬氨酸外消旋酶，我们通过定点诱变和化学修饰研究了这两种酶的半胱氨酸残基的作用。当Cys73或Cys184被Ala取代或修饰后，谷氨酸消旋酶失去了转移α质子的能力。另一方面，磷酸腺苷外消旋酶通过修饰或替换每个二聚体酶一个Cys73而完全失活。因此，我们认为天冬氨酸消旋酶可能具有复合活性位点。