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

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