APPLICATION OF 3-DIMENSIONAL HOMOLOGY MODELING OF CYTOCHROME-P450 2B1FOR INTERPRETATION OF SITE-DIRECTED MUTAGENESIS RESULTS

Three-dimensional structures of cytochrome P450 2B1 were modeled based on the crystallographic structure of P450(cam). The effect of the ali gnment, loop choice, and minimization with or without water was assess ed. Although final models were similar in overall structure, the ident ity of active site residues depended upon the alignment. An example is Phe-206, which may or may not form part of the active site. The choic e of the loop conformation had a lesser effect, while including water in the final minimization step was essential for preserving the shape and size of the active site. The best model (model 2) was in good agre ement with the data from site-directed mutagenesis studies, and correc tly predicted the effect of substitutions at 9 out of 10 amino acid po sitions. Thus, residues important for P450 2B1 activity, such as Ile-1 14, Phe-206, Ile-290, Thr-302, Val-363, and Gly-478, constitute part o f the active site and are able to interact with the substrate androste nedione through hydrophobic interactions. On the other hand, Ser-303, Ser-360 and Lys-473 are far from the active site and/or cannot interac t with the substrate, in agreement with experimental data. The model i ndicates other residues likely to be important for enzyme function, su ch as Tyr-111, Leu-209, Ile-477, and Ile-480, which can be tested expe rimentally. The substrate may assume numerous binding orientations con sistent with observed patterns of hydroxylation at C-15 and C-16. The replacement in the model of certain amino acid residues to mimic resid ue substitutions from site-directed mutagenesis studies and docking of the substrate into the modified active site allowed a plausible expla nation for alterations in regio- and stereospecificities of some mutan ts of P450 2B1, such as Gly-478 --> Ala or Val-363 --> Ala.