title: Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations.
abstract: Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH3, or -CF3 at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-α-pinene was solved at 2.2 Å and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 Å with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.
authors.affiliation: School of Pharmacy, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: Jingbao
authors.initials: J
authors.lastname: Liu
authors.affiliation: School of Pharmacy, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: Manish B
authors.initials: MB
authors.lastname: Shah
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Qinghai
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: C David
authors.initials: CD
authors.lastname: Stout
authors.affiliation: School of Pharmacy, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
authors.affiliation: School of Pharmacy, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: P Ross
authors.initials: PR
authors.lastname: Wilderman
mesh_terms: D000478:Alkylation; D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism*; D065702:Cytochrome P-450 CYP2B6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D054879:Halogenation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D008024:Ligands; D011994:Recombinant Proteins; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit)