title: Secosteroid mechanism-based inactivators and site-directed mutagenesis as probes for steroid hormone recognition by 3 alpha-hydroxysteroid dehydrogenase.
abstract: Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) inactivates circulating androgens, progestins, and glucocorticoids. 3 alpha-HSD is a member of the aldo-keto reductase superfamily, and the X-ray structure of the apoenzyme shows the presence of an (alpha/beta)8 barrel [Hoog, S. S., Pawlowski, J. E., Alzari, P. M., Penning, T. M., & Lewis, M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2517-2521]. As yet, a three-dimensional structure of the ternary complex E.NADPH.steroid is unavailable. To identify regions of the enzyme involved in steroid hormone recognition, we have employed mechanism-based inactivators and site-directed mutagenesis. (3 RS)-1,10-Seco-5 alpha-estr-1-yne-3,17 beta-diol (1) and (17 RS)- 17-hydroxy-14,15-secoandrost-4-en-15-yn-3-one (3) are secosteroids which contain latent Michael acceptors (alpha,beta-unsaturated alcohols) at opposite ends of the steroid nucleus (at the C-3 and C-17 positions, respectively). It was found that compounds 1 and 3 inactivated 3 alpha-HSD only in the presence of NAD+. The requirement for cofactor implies that 1 and 3 are oxidized to the corresponding alpha,beta-unsaturated ketones for inactivation to occur. Chemically prepared 17 beta-hydroxy-1,10-seco-5 alpha-estr-1-yn-3-one (2) and 14,15-secoandrost-4-en-15-yne-3,17-dione (4), the presumed products of 1 and 3 oxidation, behaved as stoichiometric inactivators of 3 alpha-HSD. In the presence and absence of NAD+, 2 and 4 inactivated > 50% of the enzyme in 10 s or less.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
authors.forename: B P
authors.initials: BP
authors.lastname: Schlegel
authors.forename: J E
authors.initials: JE
authors.lastname: Pawlowski
authors.forename: Y
authors.initials: Y
authors.lastname: Hu
authors.forename: D M
authors.initials: DM
authors.lastname: Scolnick
authors.forename: D F
authors.initials: DF
authors.lastname: Covey
authors.forename: T M
authors.initials: TM
authors.lastname: Penning
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D001665:Binding Sites; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D015335:Molecular Probes; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D051381:Rats; D017207:Rats, Sprague-Dawley; D012632:Secosteroids / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D015335:Molecular Probes; D012632:Secosteroids; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)