title: Functional importance of a peripheral pocket in mammalian cytochrome P450 2B enzymes.
abstract: The functional importance of a peripheral pocket found in previously published X-ray crystal structures of CYP2B4 and CYP2B6 was probed using a biophysical approach. Introduction of tryptophan within the pocket of CYP2B4 at F202 or I241 leads to marked impairment of 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) or 7-benzyloxyresorufin O-dealkylation efficiency; a similar substitution at F195, near the surface access to the pocket, does not affect these activities. The analogous CYP2B6 F202W mutant is inactive in the 7-EFC O-dealkylation assay. The stoichiometry of 7-EFC deethylation suggested that the decreased activity of F202W and I241W in CYP2B4 and lack of activity of F202W in CYP2B6 coincided with a sharp increase in the flux of reducing equivalents through the oxidase shunt to produce excess water. The results indicate that the chemical identity of residues within this peripheral pocket, but not at the mouth of the pocket, is important in substrate turnover and redox coupling, likely through effects on active site topology.
authors: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, United States.
authors: Hyun-Hee
authors: HH
authors: Jang
authors: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, United States.
authors: Jingbao
authors: J
authors: Liu
authors: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, United States.
authors: Ga-Young
authors: GY
authors: Lee
authors: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, United States.
authors: James R
authors: JR
authors: Halpert
authors: Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, United States. Electronic address: ross.wilderman@uconn.edu.
authors: P Ross
authors: PR
authors: Wilderman
mesh_terms: D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003374:Coumarins / Q000737:chemistry*; D065702:Cytochrome P-450 CYP2B6 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D006801:Humans; D020125:Mutation, Missense; D010084:Oxidation-Reduction
chemical_list: D003374:Coumarins; C056033:7-ethoxy-4-trifluoromethylcoumarin; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit)
keywords: Cytochrome P450; Monooxygenase coupling; Site-directed mutagenesis; Structure–function relationship