Bifunctional enzyme activity at the same active site: competitive inhibition kinetics with 3 alpha/20...

20 beta-Hydroxy-5 alpha-pregnan-3-one (HPO) is a competitive inhibitor of reduction by 3 alpha/20 beta-hydroxysteroid dehydrogenase (3 alpha/20 beta-HSD; E.C.1.1.1.53) of 17 beta-hydroxy-5 alpha-androstan-3-one (DHT; 3 alpha-activity; Ki = 4.6x10(-5)M), and of 6 beta-acetoxyprogesterone (6 beta-AP; 20 beta-activity; Ki = 4.34x10(-5)M). HPO and DHT inhibit affinity alkylation of 3 alpha/20 beta-HSD by 6 beta-bromoacetoxyprogesterone (6 beta-BAP). The facts that 1) enzyme 3 alpha-activity and 20 beta-activity are both competitively inhibited by HPO with practically identical Ki-values, 2) 6 beta-BAP is solely a 20 beta-activity substrate for 3 alpha/20 beta-HSD, 3) one mole of 6 beta-BAP reacts with one mole of 3 alpha/20 beta-HSD to simultaneously inactivate 3 alpha- and 20 beta-activity, and 4) inactivation of 3 alpha/20 beta-HSD by 6 beta-BAP is inhibited by DHT (a C19-steroid) or HPO (a C21-steroid), support the view that the same active site of 3 alpha/20 beta-HSD possesses both 3 alpha- and 20 beta-activity. Bifunctional activity at the same active site is considered for other steroid-specific enzymes in female mammalian reproductive systems.