Mechanisms of Flavoproteins

描述（由申请人提供）：黄素蛋白存在于整个代谢过程中，催化细胞中的各种关键步骤。在这些酶进行的反应中，有许多是劈裂未激活的碳氢键的。这是生物系统中的基本反应，由多种酶类催化。本提案的总体目标是了解黄蛋白催化碳氢键裂解反应的机制，并确定不同蛋白质折叠利用相同机制催化这些反应的程度。实验的重点是氧化碳和氧或氮之间的键的酶，转移相当于黄素辅因子的氢化物。代表不同类型底物的酶被选择用于研究：D-氨基酸氧化酶和色氨酸单加氧酶作为D-和l -氨基酸氧化酶，黄细胞色素b2作为羟基酸氧化酶，以及硝基烷烃氧化酶，这是一种新的酶，它代表了研究黄素蛋白形成碳离子的独特机会。提出了各种机制实验来阐明每种酶的碳氢键裂解步骤的细节。这些与突变分析相结合，旨在确定蛋白质折叠与机制相关的程度。这些研究的结果将为其他黄素蛋白的机制、黄素蛋白在指导黄素辅助因子的反应性中的作用以及生物系统劈裂碳氢键的一般问题提供洞见。

DESCRIPTION (provided by applicant): Flavoproteins are found throughout metabolism, catalyzing a variety of critical steps in the cell. Among the reactions which these enzymes carry out are many in which an unactivated carbon-hydrogen bond is cleaved. This is a fundamental reaction in biological systems, catalyzed by a variety of enzyme classes. The overall goal of the present proposal is to understand the mechanisms by which flavoproteins catalyze carbon-hydrogen bond cleavage reactions and to determine the extent to which different protein folds utilize the same mechanism to catalyze these reactions. The experiments focus on enzymes which oxidize bonds between carbon and either oxygen or nitrogen, transferring a hydride equivalent to the flavin cofactor. Enzymes representative of different classes of substrates have been selected for study: D-amino acid oxidase and tryptophan monooxygenase as D- and L-amino acid oxidases, flavocytochrome b2 as a hydroxy acid oxidizing enzyme, and nitroalkane oxidase, a novel enzyme which represents a unique opportunity to study carbanion formation by a flavoprotein. A variety of mechanistic experiments are proposed to elucidate the details of the carbon-hydrogen bond cleavage step by each enzyme. These are combined with mutational analyses designed to determine the extent to which the protein fold correlates with mechanism. The results of these studies will provide insight into the mechanisms of other flavoproteins, into the roles of the protein in directing the reactivity of the flavin cofactor and into the general problem of cleavage of carbon-hydrogen bonds by biological systems.