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 126.96.36.199), 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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|Zhao, Q; Abeygunawardana, C; Gittis, A G et al. (1997) Hydrogen bonding at the active site of delta 5-3-ketosteroid isomerase. Biochemistry 36:14616-26|
|Zhao, Q; Abeygunawardana, C; Mildvan, A S (1996) 13C NMR relaxation studies of backbone and side chain motion of the catalytic tyrosine residue in free and steroid-bound delta 5-3-ketosteroid isomerase. Biochemistry 35:1525-32|
|Zhao, Q; Abeygunawardana, C; Talalay, P et al. (1996) NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase. Proc Natl Acad Sci U S A 93:8220-4|
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|Zhao, Q; Li, Y K; Mildvan, A S et al. (1995) Ultraviolet spectroscopic evidence for decreased motion of the active site tyrosine residue of delta 5-3-ketosteroid isomerase by steroid binding. Biochemistry 34:6562-72|
|Austin, J C; Zhao, Q; Jordan, T et al. (1995) Ultraviolet resonance Raman spectroscopy of delta 5-3-ketosteroid isomerase revisited: substrate polarization by active-site residues. Biochemistry 34:4441-7|
|Wu, P; Li, Y K; Talalay, P et al. (1994) Characterization of the three tyrosine residues of delta 5-3-ketosteroid isomerase by time-resolved fluorescence and circular dichroism. Biochemistry 33:7415-22|
|Li, Y K; Kuliopulos, A; Mildvan, A S et al. (1993) Environments and mechanistic roles of the tyrosine residues of delta 5-3-ketosteroid isomerase. Biochemistry 32:1816-24|
|Austin, J C; Kuliopulos, A; Mildvan, A S et al. (1992) Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy. Protein Sci 1:259-70|
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