The mechanism causing the difference in kinetic properties between rat CYP2D4 and human CYP2D6 in the oxidation of dextromethorphan and bufuralol

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.