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

本研究采用X射线晶体学、定点突变和合成有机化学等方法，对ATP和NADPH依赖酶的酶促反应机理进行了研究。我们进行了三个CN键连接酶具有不同的底物特异性，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。我们还研究了两种具有不同立体专一性的托品酮还原酶：托品酮还原酶I和托品酮还原酶II，这两种还原酶都只产生彼此非对映体的托品碱或PSI-托品碱，结果如下：1.我们成功地将γ-谷氨酰半胱氨酸合成酶的表面半胱氨酸残基转化为丝氨酸残基，使其结晶。我们还合成了它的过渡态类似物抑制剂。利用抑制剂的动力学分析揭示了该酶活性中心结构的一些结构动机。2.我们结晶了两个托品酮还原酶，并通过多种同晶置换方法独立地求解了它们的三维结构。3.通过对天冬酰胺合成酶的晶体结构分析，确定了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物抑制剂，与抑制剂复合的酶的晶体结构分析正在进行中。

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.