Functional characterization of cytochrome P450 2B6 allelic variants.
Cytochrome P450 (P450) 2B6 is a hepatic enzyme of potential importance for the metabolism of clinically used drugs and environmental or abused toxicants. Genetic polymorphisms of CYP2B6 (CYP2B6*2, CYP2B6*3, CYP2B6*4, CYP2B6*5, CYP2B6*6 and CYP2B6*7; wild-type, CYP2B6*1) were found previously in white and Japanese populations. In the present study, the goal was to investigate the effects of amino acid substitutions on CYP2B6 function. Wild-type (CYP2B6.1) and all of the known variants of CYP2B6 (CYP2B6.2, CYP2B6.3, CYP2B6.4, CYP2B6.5, CYP2B6.6, and CYP2B6.7) were transiently expressed in COS-1 cells, and their 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activities were determined. The levels of the variant CYP2B6 proteins were relatively low compared with that of CYP2B6.1, although the differences were not significant. The activities of 7-ethoxy-4-trifluoromethylcoumarin O-deethylation on the basis of the CYP2B6 protein level at low (0.5 microM) and high (50 microM) substrate concentrations varied among wild-type and variant CYP2B6 proteins. All CYP2B6 enzymes showed typical Michaelis-Menten kinetics. The K(m) value of CYP2B6.6 was significantly higher than that of CYP2B6.1. Those CYP2B6 variants having a Lys262Arg substitution (CYP2B6.4, CYP2B6.6, and CYP2B6.7) showed increased values for V(max) and V(max)/K(m), whereas the kinetic parameters of CYP2B6.2 and CYP2B6.3 were not affected by the corresponding amino acid substitution. These results may mean that Lys262 in combination with other amino acid residues such as Gln172 and Arg487 is associated with the CYP2B6 function and that the genetic polymorphism of CYP2B6 leads to interindividual differences in xenobiotic metabolism.
Jinno
Hideto
H
Division of Environmental Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Tanaka-Kagawa
Toshiko
T
Ohno
Akiko
A
Makino
Yuko
Y
Matsushima
Erika
E
Hanioka
Nobumitsu
N
Ando
Masanori
M
D000483:Alleles; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D019556:COS Cells; D003374:Coumarins / Q000494:pharmacology; D065702:Cytochrome P-450 CYP2B6; D015870:Gene Expression; D014644:Genetic Variation; D000882:Haplorhini; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D009152:Mutagenicity Tests; D010089:Oxidoreductases, N-Demethylating; D010957:Plasmids; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013379:Substrate Specificity
D003374:Coumarins; D012333:RNA, Messenger; C056033:7-ethoxy-4-trifluoromethylcoumarin; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating