Testicular microsomal cytochrome P-450 for C21 steroid side chain cleavage. Spectral and binding studies.
Kinetic and binding studies were performed with a purified microsomal cytochrome P-450 from neonatal pig testis, the C21 side chain cleavage system (17 alpha-hydroxylase/C17,20-lyase). Binding of substrates and inhibitors was measured by spectral methods and by equilibrium dialysis. Kinetic data revealed that pregnenolone inhibits lyase activity with 17 alpha-hydroxypregnenolone as substrate (Ki, 0.3 microM) and that progesterone inhibits lyase activity with 17 alpha-hydroxyprogesterone (Ki, 1.5 microM); inhibition is competitive in both cases. Binding and kinetic studies revealed that Km, Ks, and Kd (Michaelis constant and dissociation constants determined by spectral and dialysis methods, respectively) are all considerably lower for the delta 5 substrates than for the corresponding delta 4 compounds. Equilibrium dialysis shows that there is a single binding site for the substrates of both activities (hydroxylase and lyase). Spectral studies revealed a lag in the development of the spectral shift produced by the addition of steroids and gave results compatible with a single active site, although this spectral evidence is not conclusive by itself. It is concluded that (i) the powerful forward competitive inhibition by pregnenolone and progesterone may be important in regulating synthesis of androgens in vivo; (ii) the porcine enzyme uses delta 5 substrates in preference to delta 4 substrates, thereby accounting for extensive use of the delta 5 pathway by pig testis in vivo; (iii) the evidence presented suggests one active site for both hydroxylase and lyase activities.
D006907:17-alpha-Hydroxypregnenolone / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000378:metabolism; D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006908:Hydroxyprogesterones / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D011284:Pregnenolone / Q000494:pharmacology; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D013057:Spectrum Analysis; D013254:Steroid 17-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism; D013552:Swine; D013737:Testis / Q000201:enzymology*
D065607:Cytochrome P-450 Enzyme Inhibitors; D006908:Hydroxyprogesterones; D006907:17-alpha-Hydroxypregnenolone; D011374:Progesterone; D011284:Pregnenolone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D013254:Steroid 17-alpha-Hydroxylase; C074514:steroid 11-alpha-hydroxylase; D000446:Aldehyde-Lyases