EPITOPE MAPPING OF CYTOCHROME P450CAM (CYP101)

Eighteen linear antigenically active sites were revealed in cytochrome P450 from Pseudomonas putida (P450cam) by hexapeptide scanning. These sites occupy about 31% of the protein sequence. Hexapeptide epitope s equences of P450cam are not found in other cytochromes P450, However, several cytochromes P450 contain shorter fragments of P450cam epitope sequences which may cause weak immune cross-reactions. P450cam antigen ic determinants are located generally at the boundaries of secondary s tructure elements. Mapping of P450cam antigenic determinants on the th ree-dimensional structure of this protein reveals 14 highly water-acce ssible antigenic sites and only 1 site (No. 322-327, QMLSGL) which is inaccessible to water. Several functionally important sites and amino acid residues of P450cam are localized within revealed linear epitopes or very close to them. These sites include substrate-binding regions, residues responsible for the putidaredoxin interaction (Arg72, Arg112 , Lys314, and Arg364), heme binding (Gln108, Arg112, Asp297, Arg299, a nd Cys357), and proton translocation (Lys178, Arg186, and Glu366). (C) 1997 Academic Press.