Abstract
We have copurified human placental 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5→4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH 2-terminal sequencing data. The affinity alkylator, 2α-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C 21 and C 19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations ( 50 1 − 20 1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity ( t 1 2 = 1.5−3.7 min ) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepi- androsterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2α-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism ( k′ 3 = 160.91/mol·s), while the alkylator inactivates isomerase by a unimolecular mechanism ( K i =0.14mM, k 3 =0.013 s −1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2α-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD + protects neither. From Dixon analysis, NADH competitively inhibits NAD + reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD + and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C 21 and C 19 isomerase activities at a second site on the same protein.
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