Isotopic Probes of Flavoprotein Oxidase Mechanisms

黄素酶催化多种代谢 重大反应。 黄素蛋白氧化酶属于 这些酶中最简单的，催化氢化物的转移 从它们的底物转化为分子氧， 过氧化氢 这里提出的研究将发展 利用稳态动力学同位素效应的方法， 快速反应动力学，以研究 黄素蛋白氧化酶，使用D-氨基酸氧化酶（DAAO）作为 模型 PI将确定V/K的pH依赖性 值和主要氘动力学同位素对 D-丙氨酸、D-丝氨酸和甘氨酸的V/K值;测定 如果观察到的最大同位素效应是内在的， 值;测量次级氘动力学同位素效应 为了确定碳氢键断裂和黄素 D-氨基酸氧化酶的还原是逐步的或协同的; 用人造黄素代替天然FAD， 不同的氧化还原电位和确定固有的同位素 方面的影响. 这些方法将用于评估 单个氨基酸的定点诱变结果 DAAO中的残留物。 它们还将适用于另外两个 黄素蛋白氧化酶、色氨酸单加氧酶和醇 氧化酶，以确定其普遍适用性。 %%% 黄素蛋白是一种主要的蛋白质，涉及广泛的 各种代谢过程。 催化剂的解析 这些酶的机制可以提供洞察 许多代谢重要酶的机制， 更复杂的黄素蛋白的机制，并进入一般 电子转移和氧活化的生物学机制。 这项研究将产生有关的机制， 黄素蛋白氧化酶使用动力学同位素效应，以检查 特定氨基酸残基在催化中的作用。 一般 对黄素蛋白碳-氢键断裂也有了解 关于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.