MECHANISMS OF FLAVOPROTEIN OXIDASES

碳氢键的断裂是最基本的 生物学中的化学反应。尽管它很重要，但这些机制 进行这种反应的酶还远没有完全完成。 明白了。这项提案关注的是被称为黄素蛋白的酶 氧化物酶，通过去除氢化物来氧化底物 等价物，并将其转移到紧密结合的黄素协因子。 氨基和羟基断裂碳氢键的机理 酸性氧化酶通常被认为与碳负离子有关。 中级的。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，这种机制可能涉及直接 氢化物转移。这项研究的目标是确定 D-氨基酸氧化酶断裂碳氢键的机理， 色氨酸单加氧酶和黄色素b2。这些方法依赖于 在很大程度上利用多重动力学同位素效应来确定 过渡态结构。此外，现场的动力学分析-- 将使用D-氨基酸氧化酶的特定突变体来确定 特定氨基酸残基在催化中的作用。硝基烷基氧化酶 将作为一种可能利用碳负离子的酶的例子进行研究 中级的。硝基丁基-FAD形成的机理 在与硝基乙烷的周转过程中将被阐明。被替换 硝化烷将被表征为底物；结果将是 与模型研究进行了比较。这些研究的结果与 黄素蛋白氧化酶将提供对其他 黄素蛋白和碳氢裂解的一般问题 生物系统形成的纽带。增加的了解和 所开发的方法将有助于开发其他药物的抑制剂 生物医学应用中的黄素蛋白氧化酶。

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.