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2O2-+2H+-歧化 02+H202，是抵御氧化损伤的主要防线 在正常新陈代谢和一些疾病状态下。这种催化作用需要 质子最终从溶液转移到活性中心释放产物 过氧化氢，并伴随着突出的产物抑制作用。这个 本提案的统一目标是阐明活动站点的功能 残留物在催化机理和产物抑制中的强调 质子转移的作用和疏水分子包膜的作用 金属周围的残留物。我们计划结构-功能研究使用 定点突变以改变活性位点附近的残基。脉搏 将使用辐解和扫描停流分光光度法来评估 在催化和X射线结晶学方面的变化，以直接结构变化。我们 还将调查这些残基在微调中的作用 氧化还原电势。将进行电位滴定以确定 突变体中点电位的还原及活性的影响 位点残留量，也将与催化速率有关的信息 以及产物抑制的程度。MnSOD的催化将被激活 利用外源提高蛋白质转移到活性部位的速度 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本征动力学势垒和 MnSOD中质子转移的热力学成分，数据将是 与其他体系中的质子转移相比。

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.