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(11) beta was inhibited by methyltrienolone in a competitive manner without exposure to light and cytochrome P-450(11) beta was photolabeled with methyltrienolone after irradiation with UV light. The addition of 11-deoxycorticosterone during photolabeling protected cytochrome P-450(11) beta from photolabeling. Photolabeled cytochrome P-450(11) beta 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(11) beta with that of cytochrome P-450cam revealed that the identified sequence corresponds to the region between the beta 3-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(11) beta which was not expected from the results of the primary sequence alignment with cytochrome P-450cam.
Ohnishi
T
T
Department of Biochemistry, Kagawa Medical School, Japan.
Miura
S
S
Ichikawa
Y
Y
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007700:Kinetics; D015741:Metribolone; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010777:Photochemistry; D051381:Rats; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D000345:Affinity Labels; D000596:Amino Acids; D015741:Metribolone; D013252:Steroid 11-beta-Hydroxylase