Isotopic Probes of Flavoprotein Oxidase Mechanisms

黄酮酶催化多种代谢反应。黄蛋白氧化酶是这些酶中最简单的一种，它催化氢化物当量从底物转移到分子氧中，形成过氧化氢。本研究将以d -氨基酸氧化酶（DAAO）为模型，利用稳态和快速反应动力学的动力学同位素效应来研究黄蛋白氧化酶的机制。PI将确定V/K值的pH依赖性以及d -丙氨酸、d -丝氨酸和甘氨酸对V/K值的一次氘动力学同位素效应；确定观测到的最大同位素效应是否为内在值；测量二次氘动力学同位素效应，以确定d -氨基酸氧化酶的碳氢键裂解和黄素还原是逐步的还是协同的；用具有不同氧化还原电位的人工黄素代替天然FAD，测定本征同位素效应。这些方法将用于评估DAAO中单个氨基酸残基的定点诱变结果。它们还将应用于另外两种黄素蛋白氧化酶，即色氨酸单加氧酶和酒精氧化酶，以确定它们的普遍适用性。黄蛋白是参与多种代谢过程的一类主要蛋白质。对这些酶的催化机制的阐明可以为了解许多代谢重要酶的机制、更复杂的黄蛋白的机制以及电子转移和氧活化的一般生物学机制提供见解。本研究将利用动力学同位素效应来检验特定氨基酸残基在催化中的作用，从而获得有关黄蛋白氧化酶机制的信息。黄蛋白碳氢键裂解的一般知识以及黄素和蝶呤依赖酶的氧活化也将产生。

Flavoenzymes catalyze a wide variety of metabolically significant reactions. Flavoprotein oxidases are among the simplest of these enzymes, catalyzing the transfer of a hydride equivalent from their substrates to molecular oxygen to form hydrogen peroxide. The research proposed here will develop methods utilizing kinetic isotope effects on steady state and rapid reaction kinetics to examine the mechanisms of flavoprotein oxidases, using D-amino acid oxidase (DAAO) as a model. The PI will determine the pH dependence of the V/K values and of the primary deuterium kinetic isotope effects on the V/K values with D-alanine, D-serine, and glycine; determine if the maximal observed isotope effects are the intrinsic values; measure secondary deuterium kinetic isotope effects in order to determine if carbon-hydrogen bond cleavage and flavin reduction in D-amino acid oxidase are stepwise or concerted; and replace the native FAD with artificial flavins with different redox potentials and determine the intrinsic isotope effects. These methods will then be used to evaluate the results of site-directed mutagenesis of individual amino acid residues in DAAO. They will also be applied to two additional flavoprotein oxidases, tryptophan monooxygenase and alcohol oxidase, to ascertain their general applicability. %%% Flavoproteins are a major class of proteins involved in a wide variety of metabolic processes. Elucidation of the catalytic mechanisms of these enzymes can provide insights into the mechanisms of a number of metabolically significant enzymes, into the mechanisms of more complex flavoproteins, and into general biological mechanisms of electron transfer and oxygen activation. This research will yield information pertaining to the mechanism of flavoprotein oxidases using kinetic isotope effects to examine the role of specific amino acid residues in catalysis. General knowledge about flavoprotein carbon-hydrogen bond cleavage as well as about oxygen activation by flavin and pterin dependent enzymes will also be generated.