title: Mechanism-based inactivation of 17 beta,20 alpha-hydroxysteroid dehydrogenase by an acetylenic secoestradiol.
abstract: 14,15-Secoestra-1,3,5(10)-trien-15-yne-3,17 beta-diol (1) is a mechanism-based inactivator of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (estradiol dehydrogenase, EC 1.1.1.62). Inactivation with alcohol 1 requires NAD-dependent enzymic oxidation and follows approximately pseudo-first-order kinetics with a limiting t1/2 of 82 min and a "Ki" of 2.0 microM at pH 9.2 and 25 degrees C. At saturating concentrations of NAD, the initial rate of inactivation is slower than in the presence of 5 microM NAD, suggesting that cofactor binding to free enzyme impedes the inactivation process. Glutathione completely protects the enzyme from inactivation at both cofactor concentrations. Inactivation with 45 microM tritiated alcohol 1 followed by dialysis and gel filtration demonstrates a covalent interaction and affords an estimated stoichiometry of 1.4 molecules of steroid per subunit (2.8 per dimer). Chemically prepared 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one (2) rapidly inactivates estradiol dehydrogenase with biphasic kinetics. From the latter phase, a Ki of 2.8 microM and a limiting t1/2 of 12 min at pH 9.2 were determined. Estradiol, NADH, and NAD all retard this latter inactivation phase. We propose that enzymatically generated ketone 2 inactivates estradiol dehydrogenase after its release from and return to the active site of free enzyme.
authors.forename: R J
authors.initials: RJ
authors.lastname: Auchus
authors.forename: D F
authors.initials: DF
authors.lastname: Covey
mesh_terms: D015089:20-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors*; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D004963:Estrenes / Q000138:chemical synthesis / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011485:Protein Binding; D012632:Secosteroids / Q000138:chemical synthesis / Q000494:pharmacology*
chemical_list: D004963:Estrenes; D007202:Indicators and Reagents; D012632:Secosteroids; D009243:NAD; C050965:14,15-secoestra-1,3,5(10)-trien-15-yne-3,17-diol; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase