Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Abstract

The last step of aldosterone biosynthesis, an 1 1p-hydroxylation followed by two 18-hydroxyIations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P-45Oll, (deoxycorticosterone (DOC) -corticosterone -18-hydroxycorticosterone -aldosterone). The 1 lp-and 18-hydroxylase activities were studied separately with a reconstituted enzymic system, using 1 l-de~xy Steroid Biochem. 33, 119-1241, were characterized for both activities (llp-and 18-hydroxylase). The value of reversible K, for the 18-hydroxylation (Ki = 5 pM for 18-vinylprogesterone and 30 pM for 18-ethynylprogesterone) is lower than that for the 1lp-hydroxylation (30 pM and 100-150 pM, respectively); the former inhibitor is stronger than the latter for both steps.The binding of substrates and inhibitors to the active site was also examined by difference absorption spectroscopy. 18-Vinylprogesterone gave rise to a type I spectrum with a K, value of 35 pM close to that of progesterone, while 18-ethynylprogesterone showed a reverse type I spectrum with a much higher K, value (140 pM). Based on these results, a hypothetical model, involving a conformational change of the enzyme for the second step, is proposed.Keywords: cytochrome ; P-45OI1,; mechanism-based inhibitors ; aldosterone biosynthesis ; 18-vinylprogesterone.Aldosterone overproduction leads to oedematous diseases and hypertension (Corvol et al., 1990). These disorders are clinically treated with spirolactones which act as antagonists at the receptor level. We developed a new approach namely by focusing on the inhibition of cytochrome P-4501,, (P-450,,,), the last enzyme in aldosterone biosynthesis. For this purpose, progesterone derivatives designed as mechanism-based inhibitors of P-4501,, were synthesized in our laboratory (Viger et al., 1988).The most potent inhibitors, tested with rat adrenal cell-free extracts, were 18-vinylprogesterone (1 8-VP) and 18-ethynylprogesterone (18-EP) (Fig. 1) which completely inhibit aldosterone production at 0.8 pM and 8 pM, respectively (Viger et al., 1989). In this study the inhibitions by 18-VP and 18-EP were studied more accurately using a bovine reconstituted enzymic P-450,,,, P-450,,, and P-450,,,,, cytochromes P-450 1 lP-18-hydroxylase, P-450 cholesterol side-chain cleavage, P-450 17a-hydro~ylase/C,,,~, lyase and P-450 aromatase.Enzyme. Steroid 1 ID-monoxygenase; cytochrome P-450, ,8 (EC 1.14.15.4). system including P-45011,, adrenodoxin and adrenodoxin reductase.P-450,,, purified from bovine adrenal cortex can catalyze the 1 lp-hydroxylation of 11 -deoxycorticosterone, the 18-hydroxylation of corticosterone as well as aldosterone formation (Fig. 2) (Wada et al., 1985;Yanagibashi et al., 1986). However two distinct forms of P-45Ol,,, encoded by two different genes exist in the bovine adrenal cortex (for a review, see Muller, 1993). Indeed two bovine P-45011, cDNAs, pc P-450 118-3 and pc P-450 llp-2 were cloned (Moroashi et al., 1987;Kirita et al., 1988). Expression into COS-7 cells revealed that both enzymes were capable of catalyzin...

11β‐Hydroxylase Activity in Recombinant Yeast Mitochondria

“…The yeast-made bovine P-450,,,, behaves qualitatively as a fully active bovine P-450, ,!,, converting 1 I -deoxycortisol and deoxycorticosterone (Tables 1 and 2). As expected, the 18-hydroxylase and aldosterone synthase activities remain at the limit of detection ( Table 2 and [42]). In conclusion, S. cerevisiae apparently possesses the necessary machinery to target an active bovine P-4501,, to mitochondria when expressed at a low level.…”

Mechanism‐Based Inactivation of Bovine Cytochrome P‐450U11β by 18‐Unsaturated Progesterone Derivatives

“…P-450,,,, bound to its substrate 11-deoxycorticosterone, was purified from bovine adrenal cortex according to the method described previously (Wada et al, 1985;Delorme et al, 1995). This preparation could be stored at -80°C for at least three months without any loss of enzyme activity in 50 mM potassium phosphate, pH 7.4, 0.1 mM EDTA, 0.1 mM dithiothreitol, 0.5% cholate, 0.2% Tween 20, and 10 pM 1 1-deoxycorticosterone.…”

“…Adrenodoxin and adrenodoxin reductase were purified from bovine adrenal cortex as described previously (Delorme et al, 1995). High concentrations of adrenodoxin and adrenodoxin reductase were obtained by ultrafiltration of purified proteins on a Centricon-10 concentrator (Amicon).…”

“…After extraction with chloroform, the products were chromatographed on silica-coated plates (eluent acetone/toluene, 1 : 1) as described previously (Delorme et al, 1995). The plates were analysed by means of an automatic TLC-linear analyzer (LB2821 Berthold).…”

“…But both are capable of catalyzing the three hydroxylations steps for the conversion of 1 1 -deoxycorticosterone to aldosterone with the same catalytic efficiency for each step. With this enzyme system, 18-VP and 18-EP were found to be potent competitive inhibitors of both activities of bovine P-450,,, during short incubations (Delorme et al, 1995). In the present study, we investigate whether these compounds behave as mechanism-based inactivators with the same reconstituted enzyme system.…”