STRUCTURAL STUDIES OF METHYL CYCLE ENZYMES

甲基环酶的结构研究

• 批准号: 3292440
• 负责人: Fusao None Takusagawa
• 金额: $ 7.23万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292440
• 关键词: Escherichia coli; X ray crystallography; computer graphics /printing; crystallization; enzyme complex; enzyme inhibitors; enzyme structure; enzyme substrate complex; hydrolase; methionine adenosyltransferase; methylation

The primary goal of this research project is the elucidation of the three-dimensional structures and enzyme mechanisms of S- Adenosylmethionine (AdoMet) synthetase and S-adenosylhomocysteine (AdoHcy) hydrolase. This will be accomplished by determining and analyzing the crystal structures of native enzymes as well as the structures of complexes between these enzymes and substrates (or inhibitors). Because of the important role that AdoMet mediated transmethylations play in regulating cellular events, the enzymes involved in these transmethylations are attractive targets for the design of potential chemotherapeutic agents. Both AdoMet synthetase and AdoHcy hydrolase seems to be key enzymes to maintain the transmethylation reactions. Prior to the rational design of inhibitors for these enzymes, the structural data which are the target of the proposed research must be obtained. Adequate amounts of AdoMet synthetase from Escherichia coli and AdoHcy hydrolase from Alcaligenes faecalis have been provided by collaborators, Dr. G.D. Markham and Professor S. Shimizu, respectively. We can routinely grow relatively large hexagonal bipyramidal crystals (approximately 1.0 mm) of AdoMet synthetase. The crystals belong to the hexagonal system with space group P6222 or P6422 and cell dimensions a=128.8, c=140.3 angstrom. One subunit (Mr=42.000, 384 amino-residues) of the tetrameric enzyme is in a crystallographic asymmetric unit. The crystals diffract to about 3.0 angstrom resolution using a conventional X-ray generator at room temperature. 3.5 angstrom resolution data of native and 3.7 angstrom resolution data of a UO2Cl2 derivative including anomalous scattering data were collected by using an area detector X-ray diffractometer at the Biotechnology Resource of the University of Virginia. A difference Patterson map calculated by using 5 angstrom resolution data clearly shows the major and minor sites of UO2. A search for another good heavy atom derivative is in progress. AdoHcy hydrolase is a hexamer composed of six identical subunits, molecular weight 48,000 daltons. Crystals with tetragonal shapes and thin plates are obtained. The tetragonal shaped crystals could be suitable for the crystallographic studies, but are slightly too small. Efforts to grow the large crystals are in progress.

该研究项目的主要目标是阐明 S-的三维结构和酶机制 腺苷甲氨酸（ADOMET）合成酶和S-腺苷同星半胱氨酸 （ADOHCY）水解酶。 这将通过确定和 分析天然酶的晶体结构以及 这些酶和底物之间的复合物结构（或 抑制剂）。 因为Adomet介导的重要作用 跨甲基化在调节细胞事件中发挥作用，这些酶是酶 参与这些转甲基是有吸引力的目标 潜在化学治疗剂的设计。 两个Adomet 合成酶和Adohcy水解酶似乎是维持的关键酶 跨甲基化反应。 在理性设计之前 这些酶的抑制剂，结构数据是 必须获得拟议研究的目标。 大肠杆菌的足够量的Adomet合成酶 来自亚藻素粪便中的Adohcy水解酶已经由 合作者G.D. Markham博士和S. Shimizu教授， 分别。 我们可以常规生长相对较大的六角形 Adomet合成酶的双锥体晶体（约1.0毫米）。 晶体属于六角形系统P6222 或p6422和细胞尺寸a = 128.8，c = 140.3 Angstrom。 一 四聚体酶的亚基（MR = 42.000，384个氨基分析） 是在晶体学不对称单元中。 晶体衍射 使用常规X射线到大约3.0 Angstrom分辨率 发电机在室温下。 3.5 Angstrom分辨率数据 UO2CL2衍生物的本机和3.7 Angstrom分辨率数据 通过使用区域收集包括异常散射数据 检测器X射线衍射仪在生物技术资源的 弗吉尼亚大学。 帕特森地图由 使用5个Angstrom分辨率数据清楚地显示了主要和次要的 UO2的站点。 寻找另一个良好的重原子衍生物是 进行中。 Adohcy水解酶是由六个组成的六聚体 相同的亚基，分子量48,000 dalton。 晶体与 获得四方形状和薄板。 四方 形状的晶体可能适用于晶体学研究， 但是太小了。 生长大晶体的努力是 进行中。