This project will continue study of the comparative structure and binding site topography of steroid-binding proteins. We will utilize: (a) affinity-labeling steroids (site directed irreversible inhibitors) and suicide substrates (site-generated irreversible inhibitors) with identification of alkylated residues and peptides that contain them, (b) determination of primary structure (amino acid sequence), so that steroid binding sites (peptides defined in (a)) can be localized and oriented, and (c) X-ray crystallography. For the steroid-interconverting enzymes, we will also: (a) determine stereospecificity of hydride transfer to cofactor, (b) evaluate spatial relationships of steroid and cofactor at the active site, and (c) identify those amino acid residues that proximate the site of the catalytic event and presumably play a role in inducing the transition state. We will study human placental estradiol 17 beta-dehydrogenase, the epimeric estradiol 17 alpha-and 17 beta- dehydrogenases from horse placenta, bovine testicular 20 alpha- hydroxysteroid dehydrogenase (all of which have been purified in this lab and are available in quantities sufficient to carry out these studies) and progesterone binding globulin of guinea pig serum supplied by Dr. Larry Kuhn. No unequivocal evidence exists as to how nature constructs a steroid binding site or the catalytic mechanism by which a steroid interconverting enzyme induces the transition state on its substrate. This project is designed to answer these questions. Our ultimate goal is to carry out X-ray crystallographic studies because we know that this method alone will give unequivocal data as to how active site amino acid residues are spatially related to each other and to the steroid and cofactor at the macromolecular binding site. We will also make wide application of affinity-labeling steroids and suicide substrates, less precise, but more readily applicable methods for elucidating binding site structure. Our basic hypothesis is that, after one has done enough studies with affinity-labeling compounds and suicide substrates with subsequent validation by X-ray crystallogaphy, these more readily applicable techniques may become adequate for at least definition of commonality in steroid binding sites from various sources.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK015708-18
Application #
3225430
Study Section
Reproductive Biology Study Section (REB)
Project Start
1977-12-01
Project End
1992-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
18
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Szendi, Zsuzsanna; Forgo, Peter; Tasi, Gyula et al. (2002) 1,5-Hydride shift in Wolff-Kishner reduction of (20R)-3beta,20, 26-trihydroxy-27-norcholest-5-en-22-one: synthetic, quantum chemical, and NMR studies. Steroids 67:31-8
Petrash, J M; Harter, T M; Murdock, G L (1997) A potential role for aldose reductase in steroid metabolism. Adv Exp Med Biol 414:465-73
O'Shea, D L; Gast, M J; Murdock, G L et al. (1994) Expression of engineered human 17 beta-estradiol dehydrogenase in a prokaryotic system. J Soc Gynecol Investig 1:143-9
Warren, J C; Murdock, G L; Ma, Y et al. (1993) Molecular cloning of testicular 20 alpha-hydroxysteroid dehydrogenase: identity with aldose reductase. Biochemistry 32:1401-6
Murdock, G L; Pineda, J; Nagorsky, N et al. (1991) Estradiol 17 beta-dehydrogenase: full enzymatic activity in the absence of zinc. Biochim Biophys Acta 1076:197-202
Pineda, J A; Murdock, G L; Watson, R J et al. (1990) Stereospecificity of hydrogen transfer between progesterone and cofactor by human placental estradiol-17 beta dehydrogenase. J Steroid Biochem Mol Biol 37:65-70
Pineda, J A; Murdock, G L; Watson, R J et al. (1989) Stereospecificity of hydrogen transfer by bovine testicular 20 alpha-hydroxysteroid dehydrogenase. J Steroid Biochem 33:1223-8