Ligand dependence of cytochrome P450c17 protection against proteolytic inactivation: structural, methodological and functional implications.
Rate constants for the subtilisin-catalyzed proteolytic inactivation of cytochrome P450c17 (CYP17), the endoplasmic reticulum membrane-bound limiting enzyme of gonadal androgen synthesis, have been determined in the absence and presence of various CYP17 ligands and correlated with fractional enzyme saturation (Y). Extrapolation to Y = 1 reveals 15.1-, 4.0- and 7.4-fold enzyme stabilization with progesterone (substrate-type ligand), testosterone (product-type ligand) and ketoconazole (imidazole-type inhibitory ligand), respectively. Structural features of ligand accommodation can therefore be monitored by the susceptibility of target enzymes to proteolysis. It is further proposed that specific protection of a membrane protein by ligand binding during proteolytic digestion may assist in the purification of that protein. Evidence is finally presented that the gonadotropin-induced rapid CYP17 down-regulation is not promoted by an elevation of steroid hormone levels.
Kühn-Velten
W N
WN
Laboratory of Biochemical Endocrinology, Department of Obstetrics and Gynecology, Heinrich Heine University, Düsseldorf, Germany.
Löhr
J B
JB
D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008297:Male; D008861:Microsomes / Q000201:enzymology; D011374:Progesterone / Q000494:pharmacology; D051381:Rats; D013254:Steroid 17-alpha-Hydroxylase; D013381:Subtilisins / Q000378:metabolism*; D013737:Testis / Q000201:enzymology; D013739:Testosterone / Q000494:pharmacology
D006063:Chorionic Gonadotropin; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D008024:Ligands; D013739:Testosterone; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D013254:Steroid 17-alpha-Hydroxylase; D013381:Subtilisins; D000446:Aldehyde-Lyases; D007654:Ketoconazole