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

Abstract

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.

An NADH-induced Conformational Change That Mediates the Sequential 3β-Hydroxysteroid Dehydrogenase/Isomerase Activities Is Supported by Affinity Labeling and the Time-dependent Activation of Isomerase

“…Finally, the isomerase-site-directed secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivated the isomerase activity with the expected first-order kinetics but inactivated the 3␤-HSD activity in an unexpected manner. As the concentration of the alkylating secosteroid increased, the rate of 3␤-HSD inactivation paradoxically decreased (8) instead of increasing in accordance with the Kitz and Wilson model of irreversible enzyme inhibition (9).…”

“…In addition to the NADH/NAD ϩ protection (5,8) and the secosteroid inactivation (8) studies discussed above, NADH (20) and pregnenolone (19) protected the same tryptic peptides (Arg-250, Lys-175) in the enzyme from affinity radioalkylation by 2␣-bromo[2Ј- …”

“…NADH is the coenzyme product of the 3␤-HSD reaction and is a potent, essential activator of isomerase activity (7). NADH, but not NAD ϩ , completely protects both the 3␤-HSD and isomerase activities from inactivation by the substrate-site affinity alkylators, 2␣-bromoacetoxyprogesterone (5) and 5,10-secoestr-4-yne-3,10,17-trione (8). Finally, the isomerase-site-directed secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivated the isomerase activity with the expected first-order kinetics but inactivated the 3␤-HSD activity in an unexpected manner.…”

The in vivo metabolism of tibolone in animal species

“…The 313-HSD enzyme catalyses the conversion of~5(1°)-313 hydroxysteroids, suchas dihydroepiandrosterone (DHEA), to 3-keto-~4 steroids, such as~4-androstenedione (~4_ dione); an essential step in the biosynthesis of steroid hormones. The dehydrogenase and~5(10)-isomerase reactions are probably catalysed at two different sites within one single protein (10). It is known that 3-keto steroids containing a~4-double bond are not substrates for the 3a-HSD or the 3/)-HSD enzymes.…”

Human Cytosolic 3α-Hydroxysteroid Dehydrogenases of the Aldo-keto Reductase Superfamily Display Significant 3β-Hydroxysteroid Dehydrogenase Activity

“…Reformation of DHT from 3␤-Diol is prevented, because 3␤-Diol is either irreversibly hydroxylated at the C-6 and/or C-7 position or is oxidized to (epi)androsterone (13-20). 3␣-Diol and 3␤-Diol, once formed, are also glucuronidated and sulfated, leading to elimination of the androstanes into the circulation and their final excretion (21,22).In humans, the irreversible NAD ϩ -dependent conversion of ⌬ 5 -3␤-hydroxysteroid into ⌬ 4 -3-ketosteroids is catalyzed by two members of the short chain dehydrogenase/reductase (SDR) family: the bifunctional 3␤-HSD/⌬ 5-4 ketosteroid isomerase (3␤-HSD/KSI) isoforms type 1 and type 2, which express 3␤-HSD and isomerase activity in a single protein (1,2,23). Besides their essential role in the formation of active steroid hormones, the 3␤-HSD/KSI isoforms also catalyze the NAD(H)-dependent in vitro interconversion of 3-keto-and 3␤-hydroxy-5␣-androstanes (24, 25).…”