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

Abstract

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 the rsult, 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-azasteroids 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.