MECHANISMS OF FLAVOPROTEIN OXIDASES

黄素蛋白氧化酶的机制

• 批准号: 6343051
• 负责人: PAUL F. FITZPATRICK
• 金额: $ 21.75万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6343051
• 关键词: active sites; aminoacid oxidase; cofactor; cytochrome b2; deuterium; enzyme mechanism; flavins; flavoproteins; monophenol monooxygenase; nonradiation isotope effect; site directed mutagenesis; stable isotope

Cleavage of carbon-hydrogen bonds is one of the most fundamental chemical reactions in biology. Despite its importance, the mechanisms by which enzymes carry out this reaction are far from being fully understood. This proposal focuses on the enzymes known as flavoprotein oxidases, which oxidize their substrates by removing a hydride equivalent and transferring it to the tightly bound flavin cofactor. The mechanism of carbon-hydrogen bond cleavage by the amino and hydroxy acid oxidases has generally been considered to involve a carbanion intermediate. However, the recent structure of D-amino acid oxidase and recent heavy isotope data suggest that the mechanisms may involve direct hydride transfer. The goal of the research here is to determine the mechanism of carbon-hydrogen bond cleavage by D-amino acid oxidase, tryptophan monooxygenase, and flavocytochrome b2. The methods rely heavily on the use of multiple kinetic isotope effects to determine transition state structure. In addition, kinetic analyses of site- specific mutants of D-amino acid oxidase will be used to determine the roles of specific amino acid residues in catalysis. Nitroalkane oxidase will be studied as an example of an enzyme likely to utilize a carbanion intermediate. The mechanism of formation of the nitrobutyl-FAD formed during turnover with nitroethane will be elucidated. Substituted nitroalkanes will be characterized as substrates; the results will be compared with model studies. The results of these studies with flavoprotein oxidases will provide insight into the mechanisms of other flavoproteins and of the general problem of cleavage of carbon-hydrogen bonds by biological systems. The increased understanding and the methods developed will be useful in developing inhibitors of other flavoproteins oxidases for biomedical applications.

碳氢键的断裂是最基本的键之一 生物学中的化学反应。尽管其重要性，但机制 酶是如何进行这种反应的还远未完全阐明 明白了。 该提案重点关注称为黄素蛋白的酶 氧化酶，通过去除氢化物来氧化其底物 等价物并将其转移到紧密结合的黄素辅因子上。 氨基和羟基断裂碳氢键的机理 通常认为酸性氧化酶涉及碳负离子 中间的。 然而，D-氨基酸氧化酶的最新结构和 最近的重同位素数据表明，该机制可能涉及直接 氢化物转移。 这里研究的目标是确定 D-氨基酸氧化酶裂解碳氢键的机制， 色氨酸单加氧酶和黄素细胞色素 b2。 方法依赖于 很大程度上依赖于使用多种动力学同位素效应来确定 过渡态结构。 此外，位点动力学分析 D-氨基酸氧化酶的特定突变体将用于确定 特定氨基酸残基在催化中的作用。 硝基烷氧化酶 将作为可能利用碳负离子的酶的例子进行研究 中间的。硝基丁基-FAD的形成机理 将阐明在与硝基乙烷的周转过程中的情况。 替补 硝基烷烃将被表征为底物；结果将是 与模型研究进行比较。 这些研究的结果与 黄素蛋白氧化酶将提供对其他机制的深入了解 黄素蛋白和碳氢裂解的一般问题 生物系统的结合。 加深的理解和 所开发的方法将有助于开发其他抑制剂 用于生物医学应用的黄素蛋白氧化酶。