title: The mechanism causing the difference in kinetic properties between rat CYP2D4 and human CYP2D6 in the oxidation of dextromethorphan and bufuralol.
abstract: The capacity to oxidize bufuralol (BF) and dextromethorphan (DEX) was compared kinetically between human CYP2D6 and four rat CYP2D (CYP2D1, -2D2, -2D3 and -2D4) isoenzymes in a yeast cell expression system. In BF 1''-hydroxylation and DEX O-demethylation, only CYP2D4 showed hook-shaped Eadie-Hofstee plots, the other four CYP2D enzymes exhibiting linear plots. In DEX N-demethylation, rat CYP2D2 did not show any detectable activity under the conditions used, whereas the other four enzymes yielded linear Eadie-Hofstee plots. To elucidate the mechanisms causing the nonlinear kinetics, four CYP2D4 mutants, CYP2D4-F109I, -V123F, -L216F and -A486F, were prepared. CYP2D4-V123F, -L216F and -A486F yielded linear or linear-like Eadie-Hofstee plots for BF 1''-hydroxylation, whereas only CYP2D4-A486F exhibited linear plots for DEX O-demethylation. The substitution of Phe-109 by isoleucine did not have any effect on the oxidative capacity of CYP2D4 for either BF or DEX. These results suggest that the introduction of phenylalanine in the active-site cavity of CYP2D4 simplifies complicated interactions between the substrates and the amino acid residues, but the mechanisms causing the simplification differ between BF and DEX.
authors.affiliation: Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan. shizuo@pharm.okayama-u.ac.jp
authors.forename: Shizuo
authors.initials: S
authors.lastname: Narimatsu
authors.forename: Daichi
authors.initials: D
authors.lastname: Kazamori
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Masuda
authors.forename: Takashi
authors.initials: T
authors.lastname: Katsu
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Funae
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Naito
authors.forename: Hironori
authors.initials: H
authors.lastname: Nakura
authors.forename: Shigeru
authors.initials: S
authors.lastname: Yamano
authors.forename: Nobumitsu
authors.initials: N
authors.lastname: Hanioka
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D003915:Dextromethorphan / Q000493:pharmacokinetics*; D004983:Ethanolamines / Q000493:pharmacokinetics*; D006801:Humans; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D004983:Ethanolamines; D011994:Recombinant Proteins; D003915:Dextromethorphan; C010831:bufuralol; D001189:Aryl Hydrocarbon Hydroxylases; C115670:Cyp2d4 protein, rat; D019389:Cytochrome P-450 CYP2D6