Functional characterization of three human cytochrome p450 2E1 variants with amino acid substitutions.
1. Cytochrome p450 (p450) 2E1 is a hepatic enzyme of importance for the metabolism of xenobiotics such as drugs and environmental toxicants. Genetic polymorphisms of CYP2E1 in 5'-flanking and coding regions have been found previously in Caucasian and Chinese populations. 2. In order to investigate the effects of amino acid substitutions on the function of CYP2E1, the enzymes of all known CYP2E1 variants in the coding region (CYP2E1.2, CYP2E1.3 and CYP2E1.4) with Arg76His, Val389Ile and Val179Ile substitutions, respectively, as well as the wild-type CYP2E1 (CYP2E1.1) were expressed in COS-1 cells, and their chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation activities were determined. 3. The protein level of CYP2E1.2 was reduced to 29% compared with that of CYP2E1.1. The profiles of the level of activity relative to CYP2E1.1 for chlorzoxazone 6-hydroxylation (300 microM substrate) and 4-nitrophenol 2-hydroxylation (150 microM substrate) were very similar. 4. Although the K(m) values were not significantly different among wild-type and variant CYP2E1s in any oxidation metabolism, the V(max) and V(max)/K(m) of CYP2E1.2 on the basis of the CYP2E1 protein level were 2.7-3.0-fold higher than those of CYP2E1.1. In contrast, the levels of CYP2E1 protein and catalytic activity of CYP2E1.3 and CYP2E1.4 were not affected by the corresponding amino acid substitutions. 5. The findings suggest that Arg76 is closely associated with the function of CYP2E1, and that the genetic polymorphism of CYP2E1 is one cause of interindividual differences in the toxicity of xenobiotics.
Hanioka
N
N
Division of Environmental Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. hanioka@nihs.go.jp
Tanaka-Kagawa
T
T
Miyata
Y
Y
Matsushima
E
E
Makino
Y
Y
Ohno
A
A
Yoda
R
R
Jinno
H
H
Ando
M
M
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002753:Chlorzoxazone / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis
D017931:DNA Primers; D018076:DNA, Complementary; D009596:Nitrophenols; D012333:RNA, Messenger; D019392:Cytochrome P-450 CYP2E1; D002753:Chlorzoxazone; C024836:4-nitrophenol