3 Beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase from bovine adrenals: mechanism of inhibition by 3-oxo-4-aza steroids and kinetic mechanism of the dehydrogenase.
Several 3-oxo-4-aza steroids (1) have been identified as inhibitors of the 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase catalyzed conversion of pregnenolone to progesterone. By kinetically decoupling the two enzyme activities isolated from bovine adrenal cortex, it has been demonstrated that inhibition by 1 occurs through interference of both activities. A preferred ordered association of substrates to the 3 beta-hydroxy-delta 5-steroid dehydrogenase in which the cofactor binds prior to steroid was determined by isotope exchange at equilibrium. With this result, the dead-end inhibition patterns of 1 with the dehydrogenase were interpreted to originate from a preferred association of inhibitor within an enzyme ternate containing NADH; this proposal is supported by data from multiple inhibition analysis indicating synergistic binding of NADH and 1. Similarly, inhibition of the 3-keto-delta 5-steroid isomerase by the 3-oxo-4-aza steroids was enhanced in the presence of the positive effector NADH. On the basis of pH profiles upon Vm, Vm/Km, and 1/Ki for both enzyme activities, inhibition is proposed to result from the structural similarity of 1 to intermediate states formed upon enzyme catalysis.
Brandt
M
M
Department of Medicinal Chemistry, Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406.
Levy
M A
MA
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors*; D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D001378:Azasteroids / Q000494:pharmacology; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007535:Isomerases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D011375:Progesterone Reductase / Q000037:antagonists & inhibitors*; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors*; D013379:Substrate Specificity
D001378:Azasteroids; D009243:NAD; D015096:3-Hydroxysteroid Dehydrogenases; D011375:Progesterone Reductase; D007535:Isomerases; D013251:Steroid Isomerases; C022660:steroid delta-isomerase