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，并为针对该酶的药物分子设计提供探针，我们开展了各种实验，取得了以下几个方面的结果。催化残基及反应机理的分析。通过X射线单晶分析，解决了突变酶K185N-NAD^+腺苷络合物的三级结构。根据该结构进行定点突变，成功地阐明了该酶催化的详细反应机理。自杀样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学。结果表明，产物腺嘌呤没有通过反应从酶蛋白中释放出来，因此推测酶没有发生周转。腺苷同型半胱氨酸酶样蛋白的研究：我们意识到AHCYL是腺苷同型半胱氨酸酶样蛋白的研究，因为AHCYL是该酶的天然突变体，所以对AHCYL的研究有助于该项目的实施。虽然有报道该蛋白不具有腺苷同型半胱氨酸酶的催化活性，但从酶学的角度还需要更详细的研究。通过对一级结构的精确比较，很明显，我们已经解决的四个催化残基和与辅酶结合有关的主要残基在蛋白质中是保守的。作为第一步，我们建立了在大肠杆菌中的表达系统，纯化了该蛋白，并开始了蛋白质化学和酶学分析。从几条实验证据中，我们提出了一种独特的结构，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.