title: Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: identification of putative substrate access regions.
abstract: To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu359-Lys373), peptide II (Leu30-Arg48), peptide III (Phe127-Arg140), and peptide IV (Arg434-Arg443). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg133, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University in Prague, Albertov 2030, 128 40 Prague, Czech Republic. hodek@natur.cuni.cz
authors.forename: Petr
authors.initials: P
authors.lastname: Hodek
authors.forename: Martin
authors.initials: M
authors.lastname: Karabec
authors.forename: Miroslav
authors.initials: M
authors.lastname: Sulc
authors.forename: Bruno
authors.initials: B
authors.lastname: Sopko
authors.forename: Stanislav
authors.initials: S
authors.lastname: Smrcek
authors.forename: Václav
authors.initials: V
authors.lastname: Martínek
authors.forename: Jirí
authors.initials: J
authors.lastname: Hudecek
authors.forename: Marie
authors.initials: M
authors.lastname: Stiborová
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives* / Q000737:chemistry; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000648:ultrastructure*; D001665:Binding Sites; D003198:Computer Simulation; D000072467:Cytochrome P450 Family 2; D008856:Microscopy, Fluorescence / Q000379:methods; D008956:Models, Chemical; D008958:Models, Molecular; D010777:Photochemistry / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: C121999:3-azidiamantane; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D000218:Adamantane