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Basic Studies on the Catalytic Reaction Mechanism of Enzymeby Organic Chemical Approaches

有机化学方法对酶催化反应机理的基础研究

基本信息

批准号：
08456060
负责人：
ODA Jun'ichi
金额：
$ 4.93万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

In this study, we have investigated enzymatic reaction mechanism of ATP and NADPH dependent enzymes by X-ray crystallography, site-directed mutagenesis, and synthetic organie chemistry. We subjected three CN-bond ligases having different substrate specificity ; glutathione synthetase, gamma-glutamylcysteine synthetase, and asparagine synthetase. We also subjected two tropinone reductases with differnt stereospecificity ; tropinone reductase I and II.Each of these reductases only produce tropine or PSI-tropine that are diastereomeric with each other.The following results were archived.1.We succeed to crystallize gamma-glutamylcysteine synthetase by alteration of its surface cysteine residues into serine residues. We also synthesized its transition state analogue inhibitors. Kinetic analysis using the inhibitiors suggested some structural motives of the active site architecture of this enzme.2.We crystallized two tropinone reductases and solved their three-dimensional structures by multiple isomorphous replacement methods independently. The results implicated the structural basis for their stereospecific reaction.3.We determine theimportant active site residues of asparagine synthetase by site-directed mutageneses of those residues that proposec from crystal structure analysis. We also succeed to synthesize its transition-state analogue inhibitor and the crystal structure analysis of the enzyme complexed with the inhibitor is in progress.

在这项研究中，我们通过x射线晶体学、定点诱变和合成有机化学研究了ATP和NADPH依赖酶的酶促反应机制。我们研究了三种具有不同底物特异性的cn键连接酶；谷胱甘肽合成酶，γ -谷氨酰半胱氨酸合成酶和天冬酰胺合成酶。我们还研究了两种立体特异性不同的托品酮还原酶；托品酮还原酶I和II。每一种还原酶只产生彼此非对映异构体的托品或psi -托品。存档了以下结果。我们通过将γ -谷氨酰半胱氨酸合成酶表面的半胱氨酸残基转化为丝氨酸残基，成功地结晶了γ -谷氨酰半胱氨酸合成酶。我们还合成了它的过渡态类似抑制剂。利用这些抑制剂进行动力学分析，揭示了该酶活性位点结构的一些结构动机。我们结晶了两种托品酮还原酶，并通过多种同构置换方法独立求解了它们的三维结构。结果揭示了它们立体特异反应的结构基础。我们通过晶体结构分析提出的位点定向突变来确定天冬酰胺合成酶的重要活性位点残基。我们还成功地合成了它的过渡态类似抑制剂，并对与该抑制剂配合的酶进行了晶体结构分析。