RAPID KINETIC AND OTHER STUDIES OF BIOLOGICAL OXYGENASES

这项计划是一个多学科的结构和功能的研究 加氧酶和其他氧化还原酶。我们的目标是了解这种化学物质 氧合酶激活氧气的机制。发现了加氧酶 在所有需氧生物体中，并在生物合成中起重要作用， 类固醇、核酸、儿茶酚胺的转化和降解， 胶原蛋白、药物、前列腺素、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。 这项提案的目的是调查四种不同类型的 我们以均一形式分离出来的加氧酶。我们会澄清 反应中的中间体并定义氨基酸残基如何影响 下列蛋白质的功能：1)黄素蛋白羟化酶，如 对羟基苯甲酸羟基酶。2)邻苯二甲酸双加氧酶，a 转化未活化芳香族化合物的多组分双加氧酶系统 将化合物转化为二氢二醇。这种类型的加氧酶在 芳香族化合物在环境中的降解，人们对此知之甚少 目前。3)与S.Ragsdale关于乙酰辅酶A的合作研究 厌氧细菌中的生物合成。该系统由一系列 具有钴胺、镍和铁硫中心的酶。他们的反应 涉及到与钴胺之间的甲基转移，非常类似于我们 用蛋氨酸合成酶测定。4)半乳糖氧化酶，一种铜 含有一种不寻常的酪氨酸基的酶。我们会调查的 该自由基参与了许多乙醇酸的氧化反应 底物。 拟议的研究将使用快速动力学分光光度法、化学 淬火等酶学方法。X射线结晶学和 基因技术，包括克隆、基因测序和突变， 将被广泛使用，以更好地理解这些 有趣的酵素。我们的方法将是修饰活性中心残基 激活底物或辅因子，然后通过上面的研究 物理技术如何在催化中的各个步骤，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有助于更好地理解 分子氧如何被激活以进行受控的代谢过程。 这反过来可能导致预测各种化合物将如何变化的能力 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.