MECHANISM OF ADENYLATE KINASE

腺苷酸激酶的机制

• 批准号: 3302300
• 负责人: MING-DAW TSAI
• 金额: $ 19.25万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302300
• 关键词: X ray crystallography; active sites; adenosine; adenosine monophosphate; adenosine triphosphate; adenosinetriphosphatase; adenylate kinase; biophysics; chemical kinetics; circular dichroism; conformation; divalent metal; enzyme mechanism; enzyme structure; enzyme substrate analog; enzyme substrate complex; intermolecular interaction; mutant; nuclear magnetic resonance spectroscopy; protein engineering; site directed mutagenesis; stereochemistry; thiophosphate

The objective of this proposal is to study the quantitative structure- function relationship of adenylate kinase (AK) by a combination of genetic, biochemical, bioorganic, and biophysical techniques. The system will be the AK from chicken muscle expressed in E. coli. The Specific Aim A is to continue the "iterative structure-function studies" to further identify catalytic residues and define their functions. The major areas of emphasis are: (a) interactions with the adenosine moiety of MgATP; (b) interactions with the adenosine moiety of AMP; (c) complementary substrate mutagenesis; (d) creating kinases with broader substrate specificity; (e) interactions with the phosphates; (f) metal ion specificity; and (g) correlation of ATPase activity and conformational changes. The Specific Aim B is to manipulate the phosphorus stereospecificity of AK. The detailed interactions between the active site residues and the phosphate groups of the substrates, and their contribution to catalysis, will be probed by using various isomers of phosphorothioates. A significant perturbation in the stereospecificity, if observed, is evidence for direct interaction between the mutated residue and the phosphate site. On the other hand, knowledge of the role of the mutated residue can lead to predictions of changes in the phosphorus stereospecificity. Specific Aim C involves total NMR assignment and structural determination of free AK, AK-AMP, AK- MgAMPPCP, AK-AMP-MgAMPPCP, and AK-MgAP5A, using enzymes uniformly labeled with 15N and/or 13C. The main purposes are to support structure-function studies in Specific Aims A and B, and to provide a detailed molecular description of conformational changes during the catalytic cycle. The Specific Aim D is to develop the use of unnatural amino acids for structure-function studies of AK. This approach will be used to complement the structure-function studies in Specific Aims A and B, with a particular emphasis on the structural and functional roles of the phosphate-binding loop (P-loop). The P-loop residues likely to be H-bonded to the phosphate moieties of substrates via the backbone NH (Gly-16, 18, 20, and 22) will be changed to the corresponding N-methyl glycine. In addition, other possible backbone interactions with the adenosine moieties will be probed accordingly. Most importantly, the aromatic ring of Tyr-95 (which is in proximity to the adenosine moiety of AMP) will be substituted with cyclohexadiene or cyclopentene to probe the structural and functional importance of the aromaticity.

该提案的目的是研究定量结构 - 通过遗传，腺苷酸激酶（AK）的功能关系 生化，生物有机和生物物理技术。 系统将是 鸡肉肌肉的AK在大肠杆菌中表达。 具体目标是 继续“迭代结构功能研究”，以进一步识别 催化残基并定义其功能。 重点的主要领域 为：（a）与MGATP的腺苷部分相互作用； （b）相互作用 带有AMP的腺苷部分； （c）互补的底物诱变； （d）创建具有更广泛的底物特异性的激酶； （e）相互作用 与磷酸盐； （f）金属离子特异性； （g） ATPase活性和构象变化。 具体目标B是 操纵AK的磷立体特异性。 详细 活性位点残基与磷酸盐组之间的相互作用 底物及其对催化的贡献将由 使用各种磷酸盐剂的异构体。 在 如果观察到的立体特定性是直接相互作用的证据 在突变的残基和磷酸盐位点之间。 另一方面， 了解突变残留物的作用可以导致预测 磷立体特异性的变化。 特定的目标c涉及总数 NMR分配和自由AK，AK-AMP，AK-的结构确定 MGAMPPCP，AK-AMP-MGAMPPCP和AK-MGAP5A，使用均匀标记的酶 具有15N和/或13C。 主要目的是支持结构功能 特定目的A和B的研究，并提供详细的分子 催化周期中构象变化的描述。 这 具体目的D是开发使用不自然氨基酸的使用 AK的结构功能研究。 这种方法将用于补充 特定目的A和B中的结构功能研究，特定于 强调磷酸结合的结构和功能作用 循环（P环）。 P环残基可能被H键成磷酸盐 通过骨干NH（Gly-16、18、20和22）的基板部分将是 改为相应的N-甲基甘氨酸。 另外，其他可能 将探测与腺苷部分的骨干相互作用 因此。 最重要的是，Tyr-95的芳香环（在 靠近AMP的腺苷部分）将用 环己二烯或环戊烯以探测结构和功能 芳香性的重要性。