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 microM secosteroid, the dehydrogenase activity is alkylated in a site-specific manner (pregnenolone slows inactivation) that follows first-order inactivation kinetics (KI = 4.2 microM, k3 = 1.31 x 10(-2) min-1). As the secosteroid level increases from 11 to 30 microM, 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 (KI = 31.3 microM, k3 = 6.42 x 10(-2) min-1) at 11-30 microM secosteroid. 5-Androstene-3,17-dione protects isomerase from inactivation by 15 microM secosteroid, but the substrate steroid unexpectedly fails to slow the inactivation of isomerase by a lower concentration of alkylator (5 microM). 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.
Thomas
J L
JL
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110.
Strickler
R C
RC
Myers
R P
RP
Covey
D F
DF
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000345:Affinity Labels; D004963:Estrenes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D010920:Placenta / Q000201:enzymology*; D011487:Protein Conformation; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D004963:Estrenes; C024783:5,10-secoestr-5-yne-3,10,17-trione; D015096:3-Hydroxysteroid Dehydrogenases; D013251:Steroid Isomerases; C022660:steroid delta-isomerase