STRUCTURAL STUDIES OF METHYL CYCLE ENZYMES

甲基环酶的结构研究

• 批准号: 2178734
• 负责人: Fusao None Takusagawa
• 金额: $ 8.87万
• 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 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178734
• 关键词: STRUCTURAL; STUDIES; METHYL; CYCLE; ENZYMES

The primary goal of this research project continues to be the elucidation of the three-dimensional structures of S-Adenosylmethionine (Adomet) synthetases from a variety of sources, including mutants. This will be accomplished by determining and analyzing the crystal structures of native enzymes as well as the structures of the complexes between these enzymes and substrates (or inhibitors). Finally, the information gained will be used to understand the functions of this important enzyme. The rational design of the potential inhibitors of the enzyme will be carried out based on the three-dimensional structural information. The structure of AdoMet synthetase crystallized in the hexagonal form has been determined at 3.0A resolution in the previous grant period. The current crystallographic R-factor is 0.32; refinement is in progress. Unfortunately, the complex between the enzyme and its substrates (ATP and L-methionine) could not be made in the hexagonal form. The preliminary study suggests that the substrates can be soaked into crystals in the tetragonal form. Therefore crystal structure analysis of the tetragonal form is in this proposal. Adequate amounts of AdoMet synthetase for this research project have been and will be provided by our collaborator, Dr. G. D. Markham. Crystallization conditions to grow relatively large tetragonal bipyramidal crystals (~0.7 nm) have been characterized in the previous grant period. The crystals belong to the tetragonal system with space group P41212 or P43212 and cell dimensions a=b=121.1, c=173.2A. Two subunits of the tetrameric enzyme are in a crystallographic asymmetric unit. The crystals diffract to about 3.5A resolution using a conventional X-ray generator at room temperature. The structure is expected to be solved with a molecular replacement method. In addition to the structure determination of the tetragonal form, the structures of several mutants will be determined in this grant period. They are low and high temperature sensitive mutants (metK 501 and 502) and a mutant in which Cys 90 and Cys 240 residues are replaced with the alanine residues. Two enzymes from new sources, a monomeric enzyme of metX gene product of E. coli and the alpha-subunit of the human enzyme, will be crystallized and their preliminary X-ray studies will be carried out.

这个研究项目的主要目标仍然是阐明 S-腺苷蛋氨酸（Adomet）的三维结构 合成酶来自各种来源，包括突变体。 这将是 通过测定和分析天然的晶体结构来完成 酶以及这些酶之间的复合物的结构 和底物（或抑制剂）。 最后，获得的信息将是 用来了解这种重要酶的功能。 合理 酶的潜在抑制剂的设计将基于 关于三维结构信息。 以六方晶形式结晶的蛋氨酸合成酶的结构具有 在上一个授予期间以3.0A决议确定。 的 目前的晶体学R因子为0.32;正在进行改进。 不幸的是，酶与其底物（ATP和 L-甲硫氨酸）不能制成六边形形式。 初步研究表明，基质可以浸泡在 呈四方形的晶体。 因此，晶体结构分析 在本发明中为四环素形式。 足量的蛋氨酸 本研究项目的合成酶已经并将由我们的 合作者G博士D.马卡姆 生长的结晶条件 相对大的四角双锥晶体（~ 0.7nm）已经被 在上一个补助期的特点。 这些晶体属于 空间群为P41212或P43212的四面体系统和晶胞尺寸 a=B=121.1，c= 173.2 A。 四聚体酶的两个亚基在一个 晶体不对称单位 晶体的电流密度约为3.5A 在室温下使用常规X射线发生器的分辨率。 的 结构有望用分子置换法解决。 除了四氢呋喃形式的结构测定外， 几个突变体的结构将在此资助期内确定。 它们是低温和高温敏感突变体（metK 501和502）， 其中Cys 90和Cys 240残基被丙氨酸取代的突变体 残基 来自新来源的两种酶，metX基因的单体酶 E.大肠杆菌和人类酶的α亚基，将是 结晶和他们的初步X射线研究将进行。