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

在本研究中，我们用X射线结晶学、定点诱变和合成有机化学的方法研究了依赖于ATP和NADPH的酶的反应机理。我们测试了三种底物专一性不同的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氨酸合成酶。我们还研究了两种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相互非对映异构体的托品或PSI托品。结果如下：1.我们成功地将γ-谷氨酰半胱氨酸合成酶表面的半胱氨酸残基转变为丝氨酸残基。我们还合成了其过渡态类似物抑制剂。通过对抑制剂的动力学分析，揭示了该酶活性部位构型的某些结构动机。2.我们用多种同象置换方法独立结晶了两个托品酮还原酶，并对它们的三维结构进行了求解。通过对晶体结构分析得到的天冬酰胺合成酶活性残基进行定点突变，确定了天冬酰胺合成酶的重要活性部位残基。我们还成功地合成了其过渡态类似物抑制剂，并对与其络合的酶进行了晶体结构分析。

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.