Active-site directed inactivation of rat ovarian 20 α-hydroxysteroid dehydrogenase

Abstract

Rat ovarian 20 alpha-hydroxysteroid dehydrogenase plays a pivotal role in leuteolysis and parturition by catalysing the reduction of progesterone to give the progestationally inactive steroid 20 alpha-hydroxyprogesterone. Putative mechanism based inhibitors of this enzyme were synthesized as potential progestational maintaining agents, including the epimeric allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol and the related vinyl ketone 1-(3 beta-hydroxy-5 alpha-androstan-17 beta-yl)-2-propen-1-one. The vinyl ketone inactivates rat ovarian 20 alpha-hydroxysteroid dehydrogenase, semi-purified by poly(L-lysine)-agarose column chromatography, in a rapid time-dependent manner. Analysis of the pseudo-first-order inactivation plots gave a Ki of 2.0 microM for the inhibitor and a t1/2 for the enzyme of 20 s at saturation. These data indicate that the vinyl ketone is a potent and efficient inactivator of the ovarian dehydrogenase. Neither dialysis in the presence or absence of a competing nucleophile nor gel filtration reserves the inactivation, suggesting that a stable covalent bond is formed between the enzyme and steroid ligand. Both substrates (20 alpha-hydroxyprogesterone and NADP+) protect the enzyme from inactivation; moreover, initial velocity measurements in the presence of saturating concentrations of both substrates indicate that the vinyl ketone can behave as a competitive inhibitor, yielding a Ki value identical with that obtained in the inactivation experiments. Our results imply that the vinyl ketone is an active-site directed alkylating agent. By contrast the allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol are neither substrates nor inhibitors of the ovarian enzyme and appear to be excluded from the catalytic site. The rapid inactivation observed with the vinyl ketone suggests that this compound may be useful as a progestational maintaining agent.

Evidence that enzyme-generated aromatic Michael acceptors covalently modify the nucleotide-binding site of 3α-hydroxysteroid dehydrogenase

“…The same phenomena may also be observed for other HSDs interacting with mechanism-based and/or affinity alkylators. For example, 3a,20/?-HSD from Streptomyces hydrogenans (Betz & Warren, 1968), and rat ovarian 20a-HSD (Pongsawasdi & Anderson, 1984) are believed to catalyse steroid transformation by ordered kinetic mechanisms with nucleotide binding first, yet both enzymes are inactivated by steroid affinity alkylators in the absence ofadded nucleotide (Ganguly & Warren, 1971;Sweet et al, 1972;Arias et al, 1973;Strickler et al, 1975;Covey et al, 1986;Ricigliano & Penning, 1986).…”

The biosynthesis of triterpenoids, steroids, and carotenoids

“…The sponge Calyx niceaensis produces a wealth of fascinating sterols, which include the cyclopropane (23S, 24S, 28R)dihydrocalysterol (1 02), the cyclopropenes 24H-isocalysterol (103), calysterol (104), and 23H-isocalysterol (105) (which together constitute up to 75% of the total steroids of this sponge), and the unique steroidal acetylenes ( 106) and (107). Each of these compounds, except the acetylene (106), became isotopically labelled when [28-14C]24-methylenecholesterol was tested as a precursor in the same sponge.lS4 Furthermore, each of the cyclopropene-containing sterols, and the acetylene (106) but not the acetylene (107), incorporated isotopic label wne'n ~~W -I J -T L ~~; -~43: "L~~=~idL'ar6caiySteroi 1 I U4-'was supplied to the sponge.…”