Active Site Mechanisms of Free Radical Metalloenzymes

描述(由申请人提供)：自由基在许多疾病中起着重要作用 和是自由基的关键催化元素 酵素。这些自由基酶包括自由基铜氧化酶，其中一种 稳定的酪氨酸-半胱氨酸二聚体蛋白质自由基与铜离子偶联形成a 金属氧化还原络合物，其独特的稳定性使其成为 研究蛋白质自由基在催化中的作用。 我们的目标是确定金属络合物在催化反应中的作用 两种自由基氧化铜酶(半乳糖氧化酶和乙二醛)的作用机制 酶)，为底物动力学研究准备替代底物 天然活性部位和突变活性部位的氧化。元素催化 步骤将使用同位素动力学、底物剖析和 温度微扰。量子力学隧道效应在自由基中的作用 这类酶的催化作用将被系统地探索。我们会 也探测在酶的再氧化过程中形成的动力学络合物 O2，并探讨蛋白质诱变对O2的影响 还原反应。 除了深入了解催化剂的周转机理外，我们还 将调查新的蛋白质自由基氧化还原位点的来源 通过辅因子生物发生研究。该项目的这一方面将需要 工程酵母表达菌株生产未加工酵母的优势 用于成熟事件的光谱分析和动力学分析的前蛋白质。这些 实验还将揭示铜向酶前的输送。 从细胞输出的分泌途径和体内形成的 活性酶复合体。

DESCRIPTION (provided by applicant): Free radicals play essential roles in many biochemical processes and are the key catalytic elements in free radical enzymes. These radical enzymes include the radical copper oxidases in which a stable tyrosine-cysteine dimer protein radical coupled to a copper ion form a metalloradical redox complex whose unique stability makes it an ideal model for investigating the role of protein free radicals in catalysis. Our goal is to define the role of the metalloradical complex in the catalytic mechanism of two radical copper oxidases (galactose oxidase and glyoxal oxidase), preparing alternative substrates for kinetic studies of substrate oxidation by both native and mutant active sites. The elementary catalytic steps will be probed using isotope kinetics, substrate profiling, and temperature perturbations. The role of quantum mechanical tunneling in radical catalysis by this class of enzymes will be systematically explored. We will also probe kinetic complexes formed during reoxidation of the enzyme by dioxygen, and explore the consequences of protein mutagenesis on the O2 reduction reaction. In addition to developing insight into the catalytic turnover mechanism, we will investigate the origin of the novel protein free radical redox site through cofactor biogenesis studies. This aspect of the project will take advantage of engineered yeast expression strains to produce unprocessed pre-protein for spectroscopic and kinetic analysis of maturation events. These experiments will also shed light on copper delivery to the pre-apoenzyme in the secretory pathway during export from the cell and in vivo formation of the active enzyme complex.