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其重要性， 酶进行这种反应的方式还远远没有完全 明白 这项建议的重点是酶称为黄素蛋白 氧化酶，其通过除去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。 氨基和羟基裂解碳氢键的机理 酸性氧化酶通常被认为涉及碳负离子 中间体 然而，最近的结构D-氨基酸氧化酶和 最近的重同位素数据表明，机制可能涉及直接 氢化物转移 本研究的目的是确定 D-氨基酸氧化酶的碳-氢键断裂机制， 色氨酸单加氧酶和黄细胞色素B2。 述方法依赖 大量使用多种动力学同位素效应来确定 过渡态结构 此外，还进行了反应动力学分析， D-氨基酸氧化酶的特异性突变体将用于确定 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 中间体形成硝基丁基-FAD的形成机制 在硝基乙烷的周转过程中将被阐明。 取代 硝基烷烃将被表征为底物;结果将是 与模型研究相比。 这些研究的结果与 黄素蛋白氧化酶将提供深入了解的机制， 黄素蛋白和一般问题的切割碳氢 生物系统的联系。 增加了解和 所开发的方法将用于开发其他抑制剂。 用于生物医学应用的黄素蛋白氧化酶。

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.