The broad and long-term objectives of this proposal are to elucidate the mechanisms of enzymatic transformations of steroids in living systems through the isolation of pertinent enzymes in pure form; characterization of their molecular properties and three-dimensional architecture; clarification of catalytic mechanisms; definition of substrate and inhibitor specificities and stereospecificities; design of active-site directed and mechanism-based specific inhibitors to modulate steroid biosynthesis and metabolism. These studies are motivated by the need to obtain a better understanding of the regulation of steroid biosynthesis and metabolism in normal and abnormal growth.
The specific aims of the current proposal focus on delta5-3-ketosteroid isomerase (EC 5.3.3.1), a unique enzyme involved in the biosynthesis of steroid hormones which has been intensively studied in our laboratory for many years. Spectroscopic methods (NMR, EPR, UV absorption and fluorescence, UV Resonance Raman, X-ray diffraction), molecular techniques (cloning of gene, sequencing, overexpression, site-specific mutagenesis), steroid ligand binding studies, and kinetic deuterium (substrate, solvent, and combined) isotope effects are being used to elucidate the catalytic mechanism, and to reveal the topological details of active and steroid binding sites. Major emphasis will be accorded to the determination of the solution structure of delta5-3-ketosteroid isomerase and its active site mutants by high resolution one- and multi-dimensional NMR spectroscopy. The catalytic mechanism will be further elucidated by identifying reaction intermediates and rate-limiting steps by spectroscopy: (a) UV Resonance Raman and 13C-NMR spectroscopy to establish the nature of the enzyme-bound steroid reaction intermediate; (b) UV absorption, resonance Raman, and fluorescence spectroscopy of steroids with suitable chromophores will be compared with simpler model compounds in solution. Kinetic isotope measurements will also be used to determine the rate-limiting step(s) of the reaction. These experiments are expected to contribute to a better understanding of the transformations of steroid hormones and lead to design of inhibitors to modulate normal or abnormal steroid function. They will also contribute to fundamental understanding of the mechanisms of enzymatic catalysis.
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