AFFINITY LABELING OF HUMAN PLACENTAL 3-BETA-HYDROXY-DELTA-5-STEROID DEHYDROGENASE AND STEROID DELTA-ISOMERASE - EVIDENCE FOR BIFUNCTIONAL CATALYSIS BY A DIFFERENT CONFORMATION OF THE SAME PROTEIN FOR EACH ENZYME-ACTIVITY

3-beta-Hydroxy-DELTA-5-steroid dehydrogenase and steroid DELTA-isomerase copurify from human placental microsomes as a single enzyme protein. The affinity-alkylating secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivates the dehydrogenase and isomerase reactions in a time-dependent manner, but which of the two activities is targeted depends on the concentration of secosteroid. At 2-5-mu-M secosteroid, the dehydrogenase activity is alkylated in a site-specific manner (pregnenolone slows inactivation) that follows first-order inactivation kinetics (K(I) = 4.2-mu-M, k3 = 1.31 X 10(-2) min-1). As the secosteroid level increases from 11 to 30-mu-M, dehydrogenase is paradoxically inactivated at progressively slower rates, and pregnenolone no longer protects against the alkylator. The inactivation of isomerase exhibits the expected first-order kinetics (K(I) = 31.3-mu-M, k3 = 6.42 X 10(-2) min-1) at 11-30-mu-M secosteroid. 5-Androstene-3,17-dione protects isomerase from inactivation by 15-mu-M secosteroid, but the substrate steroid unexpectedly fails to slow the inactivation of isomerase by a lower concentration of alkylator (5-mu-M). A shift from a dehydrogenase to an isomerase conformation in response to rising secosteroid levels explains these results. Analysis of the ligand-induced conformational change along with cofactor protection data suggests that the enzyme expresses both activities at a bifunctional catalytic site. According to this model, the protein begins the reaction sequence as 3-beta-hydroxysteroid dehydrogenase. The products of the first step (principally NADH) promote a change in protein conformation that triggers the isomerase reaction.