title: Evaluation of comparative cytochrome P450 2B4 model by photoaffinity labeling.
abstract: A homology model of rabbit CYP 2B4 was constructed on the basis of the crystallographic structure of truncated mammalian CYP 2C5/3 and bacterial soluble CYPs. To validate the CYP 2B4 homology model photoaffinity labeling was employed. Three probes (I-III) containing a photo-labile azido-group and an amino-group on opposite ends of the molecule were designed for photoaffinity labeling of the CYP 2B4 in increasing distance from the heme iron. Spectroscopic data proved probes I (the shortest) and II (a middle sized) to be coordinated with the heme iron via their amino-groups in the enzyme active center while the probe III (the longest) was not bound in this way. This binding orientation of probes I and II is in accordance with the model predicting ion-pairing of the negatively charged side chain of CYP 2B4 Asp 105 and a positively charged nitrogen located in an appropriate position in structures of probes I and II, only. The lack of heme binding of the probe III is clear from its docking into the CYP 2B4 model since no Asp 105 ion-pairing is possible. The target of photoactivated probe II, Arg 197, in a distance of about 16.5 A from the heme iron, exactly matches the position of that amino acid residue, predicted from the CYP 2B4 homology model. Moreover, using this technique, a substrate access channel has been identified. To assess the predicted substrate-binding pocket, an interaction of a specific CYP 2B4 substrate, diamantane, was examined. In "silico" docking revealed strong binding of diamantane in an orientation allowing experimentally observed C4-hydroxylation. Our homology model of CYP 2B4 is thus consistent with experimental metabolic and photoaffinity labeling data.
authors.affiliation: Charles University, Department of Biochemistry, Hlavova 2030, 128 40 Praha 2, Czech Republic. hodek@natur.cuni.cz
authors.forename: P
authors.initials: P
authors.lastname: Hodek
authors.forename: B
authors.initials: B
authors.lastname: Sopko
authors.forename: L
authors.initials: L
authors.lastname: Antonovic
authors.forename: M
authors.initials: M
authors.lastname: Sulc
authors.forename: P
authors.initials: P
authors.lastname: Novák
authors.forename: H W
authors.initials: HW
authors.lastname: Strobel
mesh_terms: D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis* / Q000737:chemistry* / Q000145:classification; D001665:Binding Sites; D003198:Computer Simulation; D000072467:Cytochrome P450 Family 2; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D007527:Isoenzymes; D019920:Photoaffinity Labels; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit)