CATALYTIC MECHANISM OF HUMAN MN SUPEROXIDE DISMUTASE

人MN超氧化物歧化酶的催化机制

• 批准号: 6335737
• 负责人: DAVID N SILVERMAN
• 金额: $ 5.38万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6335737
• 关键词: X ray crystallography; active sites; catalyst; chemical kinetics; chemical structure function; clinical research; electron transport; enzyme activity; enzyme structure; gene therapy; intermolecular interaction; manganese; nuclear magnetic resonance spectroscopy; nucleic acid sequence; oligonucleotides; polymerase chain reaction; protein structure function; pulse radiolysis; site directed mutagenesis; spectrometry; superoxide dismutase

DESCRIPTION: Human mitochondrial manganese superoxide dismutase (MnSOD) catalyzes the dismutation of the superoxide radical anion 2O2(-) + 2H+ -> O2 + H2O2. This catalysis requires cycles of both oxidation and reduction at the metal followed by proton transfers from solution to the active site to release product peroxide. Reactive oxygen species are normally and continuously produced by numerous intracellular processes, including the mitochondrial electron transport chain, and MnSOD is extremely important as a main line of defense against oxidative damage associated with the inflammatory response and postischemic reperfusion of organs. The unifying goal of this proposal is to elucidate the role of active site residues in the catalytic mechanism of human MnSOD, and to emphasize ways to enhance the catalytic activity of MnSOD. The motivation to enhance the efficiency of MnSOD is both to understand the catalysis and to introduce clinical possibilities for gene therapy. The applicant plans structure-function studies using site-directed mutagenesis to alter residues near the active site. Stopped-flow spectrophotometry and pulse radiolysis will be used to evaluate changes in catalysis and x-ray crystallography will be used to detect structural changes. The crystal structure for human MnSOD is known. Investigators have already developed an expression system and prepared wild-type human MnSOD and two significant mutants. Activation will be achieved through several strategies some of which have been useful in other enzymes, particularly the Cu, ZnSOD and carbonic anhydrase: i) enhancement of the proton transfer mechanisms by providing intra- and intermolecular shuttle groups, ii) overcoming product (peroxide) inhibition by altering the active site region through mutagenesis, and iii) enhancement of electrostatic guidance of substrate by altering the charge character near the active site.

描述：人线粒体锰超氧化物歧化酶 (MnSOD) 催化超氧自由基阴离子 2O2(-) + 2H+ -> O2 的歧化 + H2O2。 这种催化需要氧化和还原循环 金属随后质子从溶液转移到活性位点 释放产物过氧化物。 活性氧通常是 由许多细胞内过程不断产生，包括 线粒体电子传递链中，MnSOD 极其重要 对抗氧化损伤的主要防线 炎症反应和器官缺血后再灌注。 统一的 该提案的目标是阐明活性位点残基在 人类 MnSOD 的催化机制，并强调增强 MnSOD 的催化活性。 提升效率的动力 MnSOD既是为了了解催化作用，也是为了介绍临床 基因治疗的可能性。 申请人计划结构-功能 使用定点诱变来改变活性附近的残基的研究 地点。 停流分光光度法和脉冲辐射分解将用于 评估催化作用的变化，X 射线晶体学将用于 检测结构变化。 人类 MnSOD 的晶体结构是已知的。 研究人员已经开发了表达系统并准备好了 野生型人类 MnSOD 和两个重要突变体。 激活将是 通过多种策略实现，其中一些策略在其他方面很有用 酶，特别是 Cu、ZnSOD 和碳酸酐酶：i) 增强 通过提供分子内和分子间的质子转移机制 穿梭基团，ii) 通过改变 通过诱变的活性位点区域，以及iii) 增强 通过改变附近的电荷特性来静电引导基板 活性位点。