STRUCTURAL STUDIES OF METHYL CYCLE ENZYMES

甲基环酶的结构研究

• 批准号: 3292441
• 负责人: Fusao None Takusagawa
• 金额: $ 8.25万
• 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 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292441
• 关键词: Escherichia coli; S adenosylmethionine; X ray crystallography; adenosine triphosphate; crystallization; enzyme complex; enzyme inhibitors; enzyme structure; enzyme substrate complex; human tissue; isomorphous substitution; isozymes; methionine; methionine adenosyltransferase; methylation; mutant; temperature sensitive mutant

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.

该研究项目的主要目标继续是阐明 S-腺苷甲硫氨酸的三维结构（ADOMET） 来自各种来源的合成酶，包括突变体。 这将是 通过确定和分析天然的晶体结构来完成 这些酶之间的酶以及复合物的结构 和底物（或抑制剂）。 最后，获得的信息将是 用于了解这种重要酶的功能。 理性 酶的潜在抑制剂的设计将基于 关于三维结构信息。 以六边形形式结晶的Adomet合成酶的结构具有 在上一个赠款期间以3.0A的分辨率确定。 这 电流晶体学R因子为0.32;精炼正在进行中。 不幸的是，酶及其底物之间的复合物（ATP和 l-methionine）无法以六角形形式制作。 初步研究表明，底物可以浸入 四方形式的晶体。 因此 四方形式是在此提案中。 足够数量的ADOMET 该研究项目的合成酶已经并且将由我们的 合作者G. D. Markham博士。 结晶条件生长 相对较大的四方双层锥体晶体（〜0.7 nm）已经 在上一个赠款期间的特征。 晶体属于 空间群P41212或P43212和细胞尺寸的四方系统 a = b = 121.1，c = 173.2a。 四聚酶的两个亚基在A中 晶体学不对称单元。 晶体衍射至约3.5a 在室温下使用常规X射线发电机的分辨率。 这 预计结构将通过分子替代方法解决。 除了四方形式的结构测定外， 在本赠款期间将确定几个突变体的结构。 它们是低温和高温敏感突变体（METK 501和502），并且 Cys 90和Cys 240残基被丙氨酸取代的突变体 残留物。 来自新来源的两种酶，一种METX基因的单体酶 大肠杆菌和人酶的α-亚基的产物将是 将进行结晶，并将进行初步X射线研究。