CATALYTIC MECHANISM OF HUMAN MN SUPEROXIDE DISMUTASE

描述：人类线粒体锰超氧化物歧化酶(MnSOD) 催化超氧阴离子2O2(-)+2H+&GT；O2歧化 +双氧水。这种催化需要氧化和还原的循环。 接着是质子从溶液转移到活性中心的金属 释放产品过氧化氢。正常情况下，活性氧物种 由许多细胞内过程持续产生，包括 线粒体电子传递链，而MnSOD是极其重要的 与氧化损伤相关的一条主要防线 炎症反应和器官缺血后再灌流。一元化 这项建议的目的是阐明活性位点残基在 介绍了人锰超氧化物歧化酶的催化机理，并着重介绍了提高其活性的途径。 MnSOD的催化活性。提高工作效率的动力 锰超氧化物歧化酶既能了解催化作用，又能介绍临床 基因疗法的可能性。申请者规划结构-功能 利用定点突变改变活性中心附近残基的研究 地点。停流分光光度和脉冲辐解将用于 评估催化和x射线结晶学的变化将用于 检测结构变化。人锰超氧化物歧化酶的晶体结构已知。 调查人员已经开发了一种表达系统，并准备好 野生型人锰超氧化物歧化酶和两个显著突变体。激活将是 通过几种策略实现，其中一些策略在其他策略中很有用 酶，特别是铜、锌超氧化物歧化酶和碳酸氢酶：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 通过诱变的活性部位区域，以及iii)增强 改变近区电荷性质的基片静电制导 活动站点。

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.