Isotopic Probes of Flavoprotein Oxidase Mechanisms

黄素蛋白氧化酶机制的同位素探针

• 批准号: 9204698
• 负责人: Paul Fitzpatrick
• 金额: $ 26.1万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-15 至 1996-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9204698&HistoricalAwards=false
• 关键词: Isotopic; Probes; Flavoprotein; Oxidase; Mechanisms

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.

黄素酶催化多种代谢重要反应。黄素蛋白氧化酶是这些酶中最简单的酶之一，催化从底物到分子氧的氢化物当量转移形成过氧化氢。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中的动力学同位素效应来研究黄素蛋白氧化酶的作用机制。等电点将确定与D-丙氨酸、D-丝氨酸和甘氨酸的V/K值之间的pH依赖关系以及初级动力学同位素效应对V/K值的影响；确定最大的观测到的同位素效应是否是内在值；测量次级动力学同位素效应，以确定D-氨基酸氧化酶中的碳氢键断裂和黄素还原是逐步的还是协同的；并用不同氧化还原电位的人造黄素取代天然的FAD，并确定内在的同位素效应。然后，这些方法将被用于评估DAAO中单个氨基酸残基的定点突变结果。它们还将应用于另外两种黄素蛋白氧化酶，色氨酸单加氧酶和乙醇氧化酶，以确定它们的普遍适用性。黄蛋白是一类主要的蛋白质，参与多种代谢过程。阐明这些酶的催化机制可以深入了解一些代谢重要的酶的机制，更复杂的黄素蛋白的机制，以及电子转移和氧激活的一般生物学机制。这项研究将利用动力学同位素效应来研究特定氨基酸残基在催化中的作用，从而获得与黄素蛋白氧化酶机制有关的信息。此外，还将介绍黄素蛋白碳氢键断裂以及黄素和蝶呤依赖酶激活氧气的一般知识。