The long-term objectives of this proposal are to elucidate the mechanisms of enzymatic transformations of steroids in living systems through the isolation in pure form of the pertinent enzymes; characterization of their molecular properties and three-dimensional architecture; clarification of catalytic mechanisms; definition of substrate and inhibitor specificities and stereospecificities; and design of active-site-directed and mechanism-based specific inhibitors to perturb or restrain steroid biosynthesis and metabolism. As a logical extension of the availability of highly purified hydroxysteroid dehydrogenases we have used these enzymes to develop extremely sensitive systems for the analysis of steroid profiles in small samples of tissues and body fluids. These studies are motivated by the need to obtain better understanding of the regulation of steroid biosynthesis and metabolism in normal tissues and in abnormal growth.
The specific aims of this proposal are: 1. Continuation of studies designed to elucidate the detailed molecular structure, topography of the steroid-binding site, catalytic mechanism, and remarkably high catalytic efficiency of al-3-ketosteroid isomerase through the combined use of kinetic (including isotope-kinetic), NMR, EPR, X-ray and ultraviolet spectroscopy, and the application of site-specific mutagenesis of amino acid residues to modify the structure and function of the protein. 2. Development of a comprehensive system for the microanalysis of steroids through the isolation of hydroxysteroid dehydrogenases with novel and stringent specificities from microorganisms capable of growing on steroidi as their only source of carbon. Cloning and over-expression of the most useful of these enzymes will make them more widely available for analytical purposes. Development of direct enzyme reactors in which such hydroxysteroid dehydrogenases have been immobilized on solid supports, or entrapped in reverse micelles, will extend the scope of these analyses by permitting their direct application to chromatographic fractions containing organic solvents.
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