Affinity alkylation of the active site of C21 steroid side-chain cleavage cytochrome P-450 from neonatal porcine testis: a unique cysteine residue alkylated by 17.alpha.-(bromoacetoxy)progesterone

Abstract

The affinity alkylating progesterone analogue 17-(bromoacetoxy)progesterone has been used to label the active site of a microsomal cytochrome P-450 enzyme from neonatal pig testis. The enzyme causes removal of the C20 and C21 side chains from the substrates progesterone and pregnenolone by catalyzing both 17-hydroxylase and C17,20-lyase reactions, which produce the corresponding C19 steroidal precursors of testosterone. The progesterone analogue causes simultaneous inactivation of the two catalytic activities of the enzyme by a first-order kinetic process that obeys saturation kinetics. Progesterone and 17-hydroxyprogesterone each protect the enzyme inactivation. The progesterone analogue is a competitive inhibitor of the enzyme with Ki of 8.4 .mu.M and 7.8 .mu.M for progesterone and 17-hydroxyprogesterone, respectively. The enzyme inactivation and kinetic data are consistent with a theory proposing that the analogue and the two substrates compete for the same active site. The radioactive analogue 17-([14C]bromoacetoxy)progesterone causes inactivation of the enzyme with incorporation of 1.5-2.2 mol of the analogue per mole of inactivated enzyme. When this experiment is carried out in the presence of a substrate, then 0.9-1.2 mol of radioactive analogue is incorporated per mole of inactivated enzyme. The data suggest that the analogue can bind to two different sites, one of which is related to the catalytic site. Radiolabled enzyme samples, from reactions of the 14C-labeled analogue with the enzyme alone or with enzyme in the presence of a substrate, were subjected to amino acid analysis and also to tryptic digestion and peptide mapping. Amino acid analysis revealed that two cysteine residues are modified by the analogue in the absence of substrate and one cysteine is modified in the presence of substrate. Peptide mapping of the tryptic digests gave two different radiolabeled peptides from the affinity-labeled enzyme. Each of the peptides contained one cysteine residue covalently modified with the affinity label. Incorporation of radiolabel into one of the peptides was inhibited by affinity labeling in the presence of substrate. This cysteine is located in Homology Region 1 (HR1), common to other microsomal cytochromes P-450. The other labeled cysteine (whose labeling is not protected by substrate) is located at the COOH terminus, proximal to the cysteine that is believed to be involved in the binding of the heme group (fifth ligand). Since the peptide derived from radiolabeled enzyme produced from the reaction of the analogue with enzyme in the presence of a substrate contains only the COOH-terminal S-([14C]carboxymethyl)cysteine, it is concluded that a unique cysteine that is located in HR1 forms part of the active site of the enzyme and that the same active site catalyzes both 17-hydroxylase and C17,20-lyase activities.