Ligand dependence of cytochrome P450c17 protection against proteolytic inactivation: structural, methodological and...

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.