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.

本研究项目的主要目标是阐明 S-的三维结构和酶作用机制 腺苷甲硫氨酸合成酶和S-腺苷高半胱氨酸 （α-Hcy）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的结构（或 抑制剂）。 由于BMPMet介导的重要作用 转甲基化在调节细胞活动中起作用， 参与这些转甲基化的是有吸引力的目标 设计潜在的化学治疗剂。 两个均为Met 合成酶和半胱氨酸水解酶似乎是维持 转甲基化反应。 在合理设计之前， 这些酶的抑制剂，结构数据， 必须达到所提出的研究目标。 足够量的来自大肠杆菌和 来自粪产碱杆菌的HCY水解酶已由 合作者G. D博士Markham和S.清水， 分别 我们可以常规地生长相对较大的六边形 双锥晶体（约1.0 mm）的蛋氨酸合成酶。 晶体属六方晶系，空间群为P6222 或P6422，晶胞尺寸a=128.8，c=140.3埃。 一 四聚体酶的亚基（Mr=42.000，384个氨基残基） 是一个晶体学上的不对称单元。 晶体的结晶行为 到大约3.0埃的分辨率 发电机在室温下。 3.5埃分辨率数据 UO 2Cl 2衍生物的自然和3.7埃分辨率数据 包括异常散射数据的收集， 探测器X射线衍射仪在生物技术资源的 弗吉尼亚大学。 一个差帕特森地图计算， 使用5埃分辨率的数据清楚地显示了主和次 UO 2的位置 寻找另一种好的重原子衍生物是 进行中。 同型半胱氨酸水解酶是由六个氨基酸组成的六聚体， 相同的亚基分子量48,000道尔顿 晶体 获得了四边形形状和薄板。 四方 成形的晶体可适用于晶体学研究， 但是稍微太小了。 生长大晶体的努力是 进行中。