CATALYTIC MECHANISM OF HUMAN MN SUPEROXIDE DISMUTASE

描述：人线粒体锰超氧化物歧化酶（MnSOD） 催化超氧阴离子自由基2 O2（-）+2 H + -> O2歧化 + 过氧化氢 这种催化作用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 由许多细胞内过程连续产生，包括 线粒体电子传递链，MnSOD是非常重要的， 一个主要的防线，对氧化损伤有关的 炎症反应和器官缺血后再灌注。 的统一 该建议的目的是阐明活性位点残基在 人MnSOD的催化机制，并强调如何提高 MnSOD的催化活性 提高效率的动机 MnSOD是了解催化和介绍临床 基因治疗的可能性。 申请人规划结构-功能 使用定点诱变改变活性位点附近残基的研究 绝佳的价钱 停流分光光度法和脉冲辐解法将用于 评估催化和X射线晶体学的变化将被用来 检测结构变化。 人MnSOD的晶体结构是已知的。 研究人员已经开发出一种表达系统， 野生型人MnSOD和两个显著的突变体。 激活将 通过若干战略实现，其中一些战略在其他国家是有用的， 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）通过改变产物（过氧化物）的结构来克服产物（过氧化物）抑制， 活性位点区域，和iii）增强 通过改变附近的电荷特性的衬底的静电引导 活动现场。

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.