Catalytic Mechanism of Human Mn Superoxide Dismutase

描述：（申请人描述）人线粒体超氧化物歧化酶 （MnSOD）催化超氧阴离子自由基2 O2-+2H + -歧化 O2 + H2 O2，并且作为对抗氧化损伤的主要防线是重要的 在正常代谢和许多疾病状态中。这种催化作用需要 质子最终从溶液转移到活性位点以释放产物 过氧化氢，并伴随着一个突出的产品抑制。的 这一建议的统一目标是阐明活性位点的功能 在催化机理和产物抑制中的残留物强调 质子转移的作用和疏水膜的功能 金属周围的残留物。我们计划结构功能研究， 位点特异性诱变以改变活性位点附近的残基。脉冲 将使用辐解和扫描停流分光光度法来评价 催化和X射线晶体学的变化来指导结构变化。我们 还将研究这些残留物在微调的功能， 氧化还原电位将进行电位滴定，以确定 突变体的还原中点电位，并辨别活性 位点残基，也将与催化速率相关的信息 和产物抑制的程度。MnSOD的催化作用将被激活， 使用外源性的蛋白质增强蛋白质转移到活性位点的速率 质子供体和分子内穿梭基团。马库斯利率理论将是 应用于这些数据以确定内在动力学势垒， MnSOD中质子转移的热力学成分，数据将 与其他系统中的质子转移相比。

DESCRIPTION: (Applicant's Description) Human mitochondrial superoxide dismutase (MnSOD) catalyzes the dismutation of the superoxide radical anion, 2O2- + 2H+ - 02 + H202, and is important as a main line of defense against oxidative damage in normal metabolism and in a number of disease states. This catalysis requires proton transfers ultimately from solution to the active site to release product hydrogen peroxide, and is accompanied by a prominent product inhibition. The unifying goals of this proposal are to elucidate the function of active-site residues in the catalytic mechanism and in the product inhibition emphasizing the role of proton transfer and the function of an envelope of hydrophobic residues that surround the metal. We plan structure-function studies using site-specific mutagenesis to alter residues near the active site. Pulse radiolysis and scanning stopped-flow spectrophotometry will be used to evaluate changes in catalysis and x-ray crystallography to direct structural changes. We will also investigate the function of these residues in the fine tuning of the redox potential. Potentiometric titrations will be performed to determine the reduction midpoint potentials of mutants and to discern the influence of active site residues, information which will also be related to the rates of catalysis and extent of product inhibition. Catalysis by MnSOD will be activated by enhancing the rate of protein transfer to the active site using exogenous proton donors and intramolecular shuttle groups. Marcus rate theory will be applied to these data to determine the intrinsic kinetic barrier and thermodynamic components of proton transfer in MnSOD, data that will be compared with proton transfers in other systems.