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

腺苷高半胱氨酸酶作为药物设计靶标的结构-功能关系

• 批准号: 16590220
• 负责人: GOMI Tomoharu
• 金额: $ 2.24万
• 依托单位: University of Toyama (2005-2006)Toyama Medical and Pharmaceutical University (2004)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590220/
• 关键词: Adenosylhomocysteine; Adenosvlhomocysteinase; Catalytic mechanism; Suicide-like reaction; Site-specific modification; Tertiary structure; Drug target; AHCYL; NAD結合; X線解析

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.

为了更好地了解腺苷高半胱氨酸酶的反应机理，并获得针对该酶的药物分子设计的探针，我们进行了各种实验，得到了以下几点结果。催化残留物分析及反应机理。通过单独进行的x射线晶体学分析，确定了突变酶K185N-NAD^+的腺苷复合物的三级结构。基于该结构进行定点诱变，我们成功地阐明了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，产物腺嘌呤并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。我们意识到对AHCYL的研究对执行这个项目是有用的，因为AHCYL是腺苷高半胱氨酸酶样蛋白，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，但还需要从酶学角度进行更详细的研究。通过对初级结构的精确比较，我们发现四个催化残基和与辅酶结合相关的主要残基在蛋白质中都是保守的。作为第一步，我们在大肠杆菌中建立了表达系统，纯化了蛋白质，并开始了蛋白质化学和酶学分析。从几行实验证据中，我们提出了一个独特的结构，似乎反映了AHCYL的功能。

项目成果

Catalytic mechanism of S-adenosylhomocysteine hydrolase:: Roles of His 54, Asp130, Glu155, Lys185, and Asp189

• DOI: 10.1016/j.biocel.2005.06.009
• 发表时间: 2005-11-01
• 期刊: INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
• 影响因子: 4
• 作者: Yamada, T;Takata, Y;Takusagawa, F
• 通讯作者: Takusagawa, F