RAPID KINETIC AND OTHER STUDIES OF BIOLOGICAL OXYGENASES

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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.