Structure-function relationship of adenosylhomocysteinase as a target for drug design

为了更好地了解腺苷同型半胱氨酸酶的反应机理，为靶向药物的分子设计提供探针，我们进行了多方面的实验，得到了以下结果.催化残基和反应机理分析：通过X射线晶体学分析，解析了突变酶K185 N-NAD ^+的腺苷复合物的三级结构。基于结构进行定点突变，成功阐明了该酶催化的详细反应机理.类自杀反应与核苷酶活性的关系：我们分析了与NADH型酶反应引起的腺苷降解的化学计量。结果表明，反应产物腺嘌呤没有从酶蛋白中释放出来，推测酶没有发生周转.腺苷同型半胱氨酸酶样蛋白的研究：我们认识到，已经报道的腺苷同型半胱氨酸酶样蛋白AHCYL的研究对于执行本项目是有用的，因为该蛋白是该酶的天然突变体。虽然有报道称该蛋白不具有腺苷高半胱氨酸酶的催化活性，但从酶学角度需要更详细的研究。作为一级结构的精确比较的结果，很明显，我们已经解决的四个催化残基和与辅酶结合相关的主要残基在蛋白质中是保守的。作为第一步，我们在大肠杆菌中建立了表达系统。大肠杆菌，纯化蛋白质，并开始蛋白质化学和酶学分析。从几条实验证据中，我们提出了一个独特的结构，似乎反映了AHCYL的功能。

To better understand the reaction mechanism of adenosylhomocysteinase and to get the probe to the molecular design of drugs targeting this enzyme, we carried out various experiments and obtained several results as follows.1. Analyses of the catalytic residues and the reaction mechanism.By x-ray crystallographic analysis advancing separately, the tertiary structure of an adenosine complex of mutated enzyme K185N-NAD^+ was solved. Conducting site-directed mutagenesis based on the structure, we succeeded in clarifying the detailed reaction mechanism of the enzyme catalysis.2. Relationship between the suicide-like reaction and the nucleosidase activity.We analyzed the stoichiometry of adenosine degradation caused by the reaction with the NADH-type enzyme. It was revealed that the product adenine was not released from the enzyme protein through the reaction, and thus it was presumed that the turnover of the enzyme had not been occurred.3. Studies on adenosylhomocysteinase-like protein.We realized that the studies on AHCYL which had been reported as adenosylhomocysteinase-like protein was useful to execute this project, since the protein is a natural mutant of the enzyme. Although it is reported that the protein had no catalytic activity of adenosylhomocysteinase, more detailed study is needed from the enzymological point of view. As a result of precise comparison of the primary structures, it became clear that the four catalytic residues and the main residues related to the coenzyme binding, all of which we had solved, were conserved in the protein. As an initial step, we established an expression system in the E. coli, purified the protein, and started protein-chemical and enzymological analysis. From the several lines of experimental evidence, we proposed a unique structure that seems to reflect the function of AHCYL.