STRUCTURAL STUDIES OF METHYL CYCLE ENZYMES

这项研究项目的主要目标仍然是阐明 S-腺苷甲硫氨酸(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完成的 酶以及这些酶之间的络合物的结构 和底物(或抑制剂)。最后，获得的信息将是 用来理解这种重要的酶的功能。理性的人 该酶的潜在抑制剂的设计将基于 关于三维结构信息。 以六角形结晶的ADOMet合成酶的结构具有 在上一个授权期内以3.0A的分辨率确定。这个 目前的晶体R因子为0.32；正在进行精细化。 不幸的是，酶与其底物之间的复合体(ATP和 L-蛋氨酸)不能制成六角形。 初步研究表明，底物可以被浸泡到 四方结构的晶体。因此，进行了晶体结构分析。 四角形在这份提案中。适量的ADOMet 这个研究项目的合成酶已经并将由我们的 合作者，G.D.马卡姆博士。生长的结晶条件 相对较大的四方双锥晶体(~0.7 nm)已经被 这是前一次赠款期间的特点。这些水晶属于 具有P41212或P43212空间群和晶胞尺寸的四方晶系 A=b=121.1，c=173.2a。四聚体酶的两个亚基在一个 晶体不对称单位。晶体的衍射率约为3.5A 在室温下使用常规X射线发生器进行分辨率测试。这个 结构有望用分子置换方法解决。 除了确定四方晶型的结构外， 几个突变体的结构将在这个授权期内确定。 它们是低温和高温敏感突变体(metK 501和502)和 一种突变体，其中Cys 90和Cys 240残基被丙氨酸取代 残留物。两种新来源的酶--metx基因的单体酶 大肠杆菌的产物和人类酶的阿尔法亚单位，将是 晶化，并将进行初步的X射线研究。

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.