PHOTOAFFINITY-LABELING OF CYTOCHROME P-45011-BETA WITH METHYLTRIENOLONE AS A PROBE FOR THE SUBSTRATE BINDING REGION

Methyltrienolone, a synthetic steroid, was used as a photoaffinity ligand for steroid-binding proteins. The enzymatic activity of bovine adrenocortical cytochrome P-450(11beta) was inhibited by methyltrienolone in a competitive manner without exposure to light and cytochrome P-450(11beta) was photolabeled with methyltrienolone after irradiation with UV light. The addition of 11-deoxycorticosterone during photolabeling protected cytochrome P-450(11beta) from photolabeling. Photolabeled cytochrome P-450(11beta) was digested with TPCK-treated trypsin and the peptide fragments were separated with a reverse-phase HPLC system. The labeled peptide was analyzed and its amino acid sequence was determined to be Trp428-Leu429-Asp430-Arg431 . Alignment of the primary structure of cytochrome P-450(11beta) with that of cytochrome P-450cam revealed that the identified sequence corresponds to the region between the beta3-sheet and L-helix of cytochrome P-450cam. This region of mammalian cytochromes P-450 shows poor homology with that of cytochrome P-450cam, but is well-conserved, especially at Trp-428 and preceding amino acids, as the aromatic region. The present results demonstrate that the labeled sequence contributes in part to the formation of the substrate binding pocket of cytochrome P-450(11beta) which was not expected from the results of the primary sequence alignment with cytochrome P450cam.