RAPID KINETIC AND OTHER STUDIES OF BIOLOGICAL OXYGENASES

本项目是一个多学科的结构和功能的研究， 加氧酶和其它氧化还原酶。 我们的目标是了解 氧被加氧酶激活的机制。 氧合酶被发现 在所有需氧生物中并且在生物合成中是重要的， 转化和降解类固醇，核酸，儿茶酚胺， 胶原蛋白、药物、木脂素、木质素和各种外来化合物。 这些酶对大多数生命形式至关重要。 本提案的目的是调查四种不同类型的 加氧酶，我们已经以同质形式分离。 我们将阐明 反应中的中间体，并定义氨基酸残基如何影响 以下蛋白质的功能：1）黄素蛋白羟化酶， 对羟基苯甲酸羟化酶2)邻苯二甲酸双加氧酶a 多组分双加氧酶系统，其将未活化的芳族化合物 化合物转化为二氢二醇。 这种类型的加氧酶在 芳香族化合物的环境降解， 目前。3)与S. Ragsdale使用乙酰辅酶A 厌氧细菌的生物合成。 该系统由一系列 含有钴胺素、镍和铁硫中心的酶。的反应 涉及甲基转移到钴胺素和从钴胺素，非常类似于那些我们 用甲硫氨酸合成酶测定。4)半乳糖氧化酶，一种铜 含有一种不寻常的酪氨酸基的酶。 我们将调查 这种自由基参与许多乙醇酸的氧化 印刷受体. 拟议的研究将采用快速动力学分光光度法，化学 猝灭和其它酶学方法。 x射线晶体学和 遗传技术，包括克隆、基因测序和诱变， 将被广泛使用，以更好地了解这些 有趣的酶 我们的方法是修饰活性位点残基 激活底物或辅因子，然后通过上述研究 物理技术如何在催化的各个步骤，包括形成和 中间体的反应性受到影响。 我们希望这些研究的结果将导致更好的理解 分子氧是如何被激活来控制代谢过程的 这反过来可能导致预测各种化合物将如何 在环境中代谢。

This project is a multi-disciplinary study of the structure and function of oxygenases and other redox enzymes. Our goal is to understand the chemical mechanism whereby oxygen is activated by oxygenases. Oxygenases are found in all aerobic organisms and are important in the biosynthesis, transformation, and degradation of steroids, nucleic acids, catecholamines, collagen, drugs, prostaglandins, lignin, and various foreign compounds. These enzymes are crucial to a majority of life forms. The aims of this proposal are to investigate four different types of oxygenases which we have isolated in homogeneous form. We will elucidate intermediates in the reactions and define how amino acid residues affect functions of the following proteins: 1) Flavoprotein hydroxylases such as para-hydroxybenzoate hydroxylase. 2) Phthalate dioxygenase, a multicomponent dioxygenase system which converts an unactivated aromatic compound to a dihydrodiol. This type of oxygenase is very important in environmental degradation of aromatic compounds, and is poorly understood at present. 3) A collaborative study with S. Ragsdale on acetyl-CoA biosynthesis in anaerobic bacteria. This system consists of a series of enzymes with cobalamin, nickel, and iron-sulfur centers. The reactions involve methyl transfers to and from cobalamin, very similar to those we have measured with methionine synthase. 4) Galactose oxidase, a copper containing enzyme with an unusual tyrosine radical. We will investigate the participation of this radical in the oxidation of numerous glycolic substrates. The proposed study will employ rapid kinetics spectrophotometry, chemical quenching, and other enzymological methods. X-ray crystallography and genetic techniques, including cloning, gene sequencing and mutagenesis, will be used extensively to develop a better understanding of these interesting enzymes. Our approach will be to modify active site residues that activate the substrate or cofactors and then to study by the above physical techniques how various steps in catalysis, including formation and reactivity of intermediates, are affected. We hope that results from these studies will lead to a better understanding of how molecular oxygen is activated for controlled metabolic processes. This in turn may lead to the ability to predict how various compounds will be metabolized in the environment.