X-RAY STUDIES OF PHOSPHOLIPASE A2

磷脂酶 A2 的 X 射线研究

• 批准号: 2187095
• 负责人: MUTTAIYA SUNDARALINGAM
• 金额: $ 6.25万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187095
• 关键词: X ray crystallography; active sites; chemical binding; computer data analysis; enzyme complex; enzyme inhibitors; enzyme structure; molecular site; mutant; pancreas enzyme; phospholipase A2; protein structure function; structural biology; supercomputer

The long-term objective of this proposal is to establish the structure- activity relationships of phospholipase A2 (PLA2) through the determination of the x-ray structure of its mutants and their complexes with the transition state analogue, diC8(2Ph)PE. This will be carried out in collaboration with Ming-Daw Tsai who has cloned and over expressed the enzyme and is studying the kinetics and solution structure by NMR and Michael H. Gelb who will be supplying the transition state analogue. Although, the x-ray structures of PLA2 from mammalian pancreas, snake and bee venom, synovial fluids and their complexes with the transition state analogues are known, there still are major gaps in the PLA2 structural field. For example, the absence of a systematic study of the effects of mutations on the catalytic site, lipid binding loci and interfacial recognition surface are lacking. Also there is no structural information on the effect of mutations on the binding of substrate analogues. This proposal seeks to bridge these gaps. It is planned to carry out the x- ray structures of the (i) mutants of the catalytic site (H48N, D99N, Y52F, F22X, F106X and Y69X), (ii) the mutants of the hydrophobic channel (L2X, F5X, I9X, L19X), (iii) complexes of the mutants with the transition state analogue, diC8(2Ph)PE, (iv) the mutants of the important disulfide bridges where the Cys/Cys pair is changed to Ala/Ala and (v) the mutants of the N-terminal Ala-1 for its effect on the stability of the catalytic pocket and its role in the interfacial recognition. To date the structures of the wild type (1.8 alpha), the single mutant K56M (1.8 alpha) and the double mutant Y52F/Y73F (1.93 alpha) have been solved in our laboratory. The K56M structure has indicated a possible head-group binding site for the phospholipids. The double mutant Y52F/Y73F has unequivocally established that the phenolic groups of the highly conserved Tyr residues (52 and 73) are not replaced by water molecules in the active site, and further demonstrated that the hydrogen bonding by the phenolic groups are not essential for supporting the catalytic pocket. This work is health related in that it will be useful in rational design of more potent inhibitors of PLA2 which have therapeutic applications.

该提案的长期目标是建立以下结构： 磷脂酶 A2 (PLA2) 的活性关系 其突变体及其复合物的X射线结构的测定 与过渡态类似物 diC8(2Ph)PE。 这将被携带 与已克隆并过度表达的 Ming-Daw Tsai 合作 酶并通过 NMR 研究动力学和溶液结构 Michael H. Gelb 将提供过渡态类似物。 尽管哺乳动物胰腺、蛇和哺乳动物的 PLA2 的 X 射线结构 蜂毒、滑液及其过渡态复合物 尽管PLA2的类似物已知，但PLA2结构仍存在重大差距 场地。 例如，缺乏对影响的系统研究。 催化位点、脂质结合位点和界面上的突变 缺乏识别表面。 也没有结构信息 突变对底物类似物结合的影响。 这 提案旨在弥合这些差距。 计划进行 x (i) 催化位点突变体（H48N、D99N、 Y52F、F22X、F106X 和 Y69X)，(ii) 疏水通道的突变体 (L2X, F5X, I9X, L19X), (iii) 突变体与转变的复合物 状态类似物，diC8(2Ph)PE，(iv) 重要二硫键的突变体 Cys/Cys 对变为 Ala/Ala 的桥和 (v) 突变体 N-末端Ala-1对催化稳定性的影响 口袋及其在界面识别中的作用。 迄今为止 野生型（1.8 α）的结构，单突变体 K56M（1.8 α）和双突变体 Y52F/Y73F（1.93 α）已在 我们的实验室。 K56M 结构表明了可能的头组 磷脂的结合位点。 双突变体Y52F/Y73F具有 明确地确定，高度的酚基团 保守的 Tyr 残基（52 和 73）不被水分子取代 位于活性位点，并进一步证明氢键 酚基团对于支持催化作用不是必需的 口袋。 这项工作与健康相关，因为它将有助于 合理设计更有效的 PLA2 抑制剂，具有治疗作用 应用程序。