Catalytic roles of CYP2D6.10 and CYP2D6.36 enzymes in mexiletine metabolism: in vitro functional analysis of recombinant proteins expressed in Saccharomyces cerevisiae.
Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one-third of the medicines in current clinical use and exhibits genetic polymorphism with interindividual differences in metabolic activity. To precisely investigate the effect of CYP2D6*10B and CYP2D6*36 frequently found in Oriental populations on mexiletine metabolism in vitro, CYP2D6 proteins of wild-type (CYP2D6.1) and variants (CYP2D6.10 and CYP2D6.36) were heterologously expressed in yeast cells and their mexiletine p- and 2-methyl hydroxylation activities were determined. Both variant CYP2D6 enzymes showed a drastic reduction of CYP2D6 holo- and apoproteins compared with those of CYP2D6.1. Mexiletine p- and 2-methyl hydroxylation activities on the basis of the microsomal protein level at the single substrate concentration (100 microM) of variant CYP2D6s were less than 6% for CYP2D6.10 and 1% for CYP2D6.36 of those of CYP2D6.1. Kinetic analysis for mexiletine hydroxylation revealed that the affinity toward mexiletine of CYP2D6.10 and CYP2D6.36 was reduced by amino acid substitutions. The Vmax and Vmax/Km values of CYP2D6.10 on the basis of the microsomal protein level were reduced to less than 10% of those of CYP2D6.1, whereas the values on the basis of functional CYP2D6 level were comparable to those of CYP2D6.1. Although it was impossible to estimate the kinetic parameters for the mexiletine hydroxylation of CYP2D6.36, the metabolic ability toward mexiletine was considered to be poorer not only than that of CYP2D6.1 but also than that of CYP2D6.10. The same tendency was also observed in kinetic analysis for bufuralol 1''-hydroxylation as a representative CYP2D6 probe. These findings suggest that CYP2D6*36 has a more drastic impact on mexiletine metabolism than CYP2D6*10.
Hanioka
Nobumitsu
N
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan.
Okumura
Yoshihito
Y
Saito
Yoshiro
Y
Hichiya
Hiroyuki
H
Soyama
Akiko
A
Saito
Keita
K
Ueno
Kazuyuki
K
Sawada
Jun-ichi
J
Narimatsu
Shizuo
S
D000889:Anti-Arrhythmia Agents / Q000378:metabolism*; D044466:Asian People / Q000235:genetics; D002384:Catalysis; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D014644:Genetic Variation; D006239:Haplotypes; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008801:Mexiletine / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014162:Transfection
D000889:Anti-Arrhythmia Agents; D011994:Recombinant Proteins; D008801:Mexiletine; D019389:Cytochrome P-450 CYP2D6