Physiological 3 beta-hydroxy-5-ene steroid substrates bind to 3 beta-hydroxysteroid dehydrogenase without the prior binding of cofactor.

3 beta-Hydroxy-delta 5-steroid dehydrogenase (3 beta-HSD)/steroid delta 5-4-isomerase catalyses the conversion of 3 beta-hydroxy-5-ene steroids (e.g. pregnenolone) to 3-oxo-4-ene-steroids (progesterone) in human placenta. Isotope exchange at equilibrium using NAD+/NADH and the 5 alpha-reduced steroids, 5 alpha-androstane-3 beta, 17 beta-diol and 5 alpha-androstan-17 beta-ol-3-one, determined a cofactor-first order of binding for these 3 beta-HSD substrates [1]. Exchange at equilibrium cannot be performed with 3 beta-hydroxy-5-ene steroids because 3 beta-HSD is not reversible with the 5-ene substrates. To compare their cofactor requirements for binding, 3 beta-hydroxy-5-ene and 3 beta-hydroxy-5 alpha-reduced steroids were tested as protectors against the inactivation of purified human placental 3 beta-HSD by 2 alpha-bromoacetoxyprogesterone (2 alpha-BAP) in the presence or absence of cofactor. In incubations without cofactor, pregnenolone or dehydroepiandrosterone dramatically slowed (protected) the rate of 3 beta-HSD inactivation by 2 alpha-BAP, an affinity alkylator that binds specifically at the 3 beta-HSD substrate site. In contrast, 5 alpha-androstan-3 alpha-ol-17-one, 5 alpha-androstane-3 beta, 17 beta-diol, or 11 alpha-acetoxy-5 alpha-pregnan-3,20-dione protected 3 beta-HSD from inactivation by 2 alpha-BAP only in the presence of NADH (0.3 microM) or NAD+ (10 microM). At these low concentrations, neither NADH nor NAD+ slowed the inactivation of 3 beta-HSD by 2 alpha-BAP in the absence of protector-steroid. Further, the 3-oxo-5 alpha-reduced alkylator, 11 alpha-bromoacetoxy-5 alpha-pregnan-3,20-dione (11 alpha-BA-5 alpha-P), did not inactivate 3 beta-HSD in a specific manner. After pre-incubation with NAD+ (10 microM), 11 alpha-BA-5 alpha-P inactivated 3 beta-HSD rapidly and specifically (t1/2 = 3.7 min). 11 alpha-Bromoacetoxyprogesterone inactivated 3 beta-HSD at the same rate (t1/2 = 5.0 min) in the presence or absence of NAD+. These affinity labelling studies confirm the cofactor-first binding order for 3 beta-hydroxy-5 alpha-reduced steroids, and conclusively show that the more important, physiological 3 beta-hydroxy-5-ene substrates bind to 3 beta-HSD without a cofactor requirement.