Active Site Mechanisms of Free Radical Metalloenzymes

自由基金属酶的活性位点机制

• 批准号: 6430566
• 负责人: JAMES W WHITTAKER
• 金额: $ 24.7万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6430566
• 关键词: Saccharomyces cerevisiae; X ray crystallography; active sites; alcohol oxidoreductases; cofactor; enzyme activity; enzyme mechanism; free radicals; fungal genetics; fungal proteins; galactose oxidase; metalloenzyme; mutant; site directed mutagenesis

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.

描述（由申请人提供）：自由基在许多疾病中起着重要作用。 生物化学过程，是自由基的关键催化元素 内切酶这些自由基酶包括自由基铜氧化酶，其中 稳定酪氨酸-半胱氨酸二聚体蛋白自由基与铜离子偶联形成 金属自由基氧化还原络合物，其独特的稳定性使其成为理想的模型， 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自由基络合物在催化反应中的作用。 两种自由基铜氧化酶（半乳糖氧化酶和乙二醛）的作用机制 氧化酶），制备用于底物动力学研究的替代底物 氧化的天然和突变的活性位点。元素催化 将使用同位素动力学、底物分析和 温度扰动量子力学隧穿在自由基中的作用 这类酶的催化作用将被系统地研究。我们将 还探测在酶的再氧化过程中形成的动力学复合物， 分子氧，并探讨蛋白质突变对O2 还原反应 除了深入了解催化周转机制，我们 将研究新的蛋白质自由基氧化还原位点的起源 通过辅因子生物合成研究。该项目的这一方面将采取 工程酵母表达菌株生产未加工的 用于成熟事件的光谱和动力学分析的前蛋白。这些 实验还将阐明铜在细胞中向前脱辅基酶的传递。 在从细胞输出和在体内形成的分泌途径， 活性酶复合物