Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes.
Cytochrome P450 2E1 (CYP2E1) is an enzyme of major toxicological interest because it metabolizes various drugs, precarcinogens and solvents to reactive metabolites. In this study, human and cynomolgus monkey CYP2E1 cDNAs (humCYP2E1 and monCYP2E1, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate CYP2E1s. The enzymatic properties of CYP2E1 proteins were characterized by kinetic analysis of chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation. humCYP2E1 and monCYP2E1 enzymes showed 94.3% identity in their amino acid sequences. The functional CYP content in yeast cell microsomes expressing humCYP2E1 was 38.4 pmol/mg protein. The level of monCYP2E1 was 42.7% of that of humCYP2E1, although no significant differences were statistically observed. The K(m) values of microsomes from human livers and yeast cells expressing humCYP2E1 for CYP2E1-dependent oxidation were 822 and 627 microM for chlorzoxazone 6-hydroxylation, and 422 and 514 microM for 4-nitrophenol 2-hydroxylation, respectively. The K(m) values of microsomes from cynomolgus monkey livers and yeast cells expressing monCYP2E1 were not significantly different from those of humans in any enzyme source. V(max) and V(max)/K(m) values of human liver microsomes for CYP2E1-dependent oxidation were 909 pmol/min/mg protein and 1250 nl/min/mg protein for chlorzoxazone 6-hydroxylation, and 1250 pmol/min/mg protein and 2990 nl/min/mg protein for 4-nitrophenol 2-hydroxylation, respectively. The kinetic parameter values of cynomolgus monkey livers were comparable to or lower than those of human liver microsomes (49.5-102%). In yeast cell microsomes expressing humCYP2E1, V(max) and V(max)/K(m) values for CYP2E1-dependent oxidation on the basis of CYP holoprotein level were 170 pmol/min/pmol CYP and 272 nl/min/pmol CYP for chlorzoxazone 6-hydroxylation, and 139 pmol/min/pmol CYP and 277 nl/min/pmol CYP for 4-nitrophenol 2-hydroxylation, respectively, and the kinetic parameters of monCYP2E1 exhibited similar values. These findings suggest that human and cynomolgus monkey CYP2E1 enzymes have high homology in their amino acid sequences, and that their enzymatic properties are considerably similar. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.
Hanioka
Nobumitsu
N
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan. hanioka@pharm.okayama-u.ac.jp
Yamamoto
Maki
M
Iwabu
Hiroyuki
H
Jinno
Hideto
H
Tanaka-Kagawa
Toshiko
T
Naito
Shinsaku
S
Shimizu
Takefumi
T
Masuda
Kazufumi
K
Katsu
Takashi
T
Narimatsu
Shizuo
S
D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001483:Base Sequence; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D020035:Holoenzymes / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008252:Macaca fascicularis; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008875:Middle Aged; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001051:Apoenzymes; D020035:Holoenzymes; D011994:Recombinant Proteins; D019392:Cytochrome P-450 CYP2E1