Hanioka Nobumitsu, Yamamoto Maki, Iwabu Hiroyuki, Jinno Hideto, Tanaka-Kagawa Toshiko, Naito Shinsaku, Shimizu Takefumi, Masuda Kazufumi, Katsu Takashi, Narimatsu Shizuo
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan.
Life Sci. 2007 Oct 27;81(19-20):1436-45. doi: 10.1016/j.lfs.2007.09.002. Epub 2007 Sep 15.
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.
细胞色素P450 2E1（CYP2E1）是一种具有重大毒理学意义的酶，因为它可将多种药物、前致癌物和溶剂代谢为活性代谢物。在本研究中，克隆了人及食蟹猴的CYP2E1 cDNA（分别为humCYP2E1和monCYP2E1），并在酵母细胞中异源表达相应蛋白，以鉴定灵长类CYP2E1的功能。通过对氯唑沙宗6-羟基化和4-硝基苯酚2-羟基化的动力学分析来表征CYP2E1蛋白的酶学性质。humCYP2E1和monCYP2E1酶的氨基酸序列一致性为94.3%。表达humCYP2E1的酵母细胞微粒体中的功能性CYP含量为38.4 pmol/mg蛋白。monCYP2E1的水平为humCYP2E1的42.7%，尽管未观察到统计学上的显著差异。人肝脏微粒体和表达humCYP2E1的酵母细胞微粒体对CYP2E1依赖性氧化反应中，氯唑沙宗6-羟基化的K(m)值分别为822和627 microM，4-硝基苯酚2-羟基化的K(m)值分别为422和514 microM。食蟹猴肝脏微粒体和表达monCYP2E1的酵母细胞微粒体的K(m)值在任何酶源中与人类的均无显著差异。人肝脏微粒体对CYP2E1依赖性氧化反应的V(max)和V(max)/K(m)值，氯唑沙宗6-羟基化分别为909 pmol/min/mg蛋白和1250 nl/min/mg蛋白，4-硝基苯酚2-羟基化分别为1250 pmol/min/mg蛋白和2990 nl/min/mg蛋白。食蟹猴肝脏的动力学参数值与人类肝脏微粒体相当或更低（49.5 - 102%）。在表达humCYP2E1的酵母细胞微粒体中，基于CYP全蛋白水平的CYP2E1依赖性氧化反应的V(max)和V(max)/K(m)值，氯唑沙宗6-羟基化分别为170 pmol/min/pmol CYP和272 nl/min/pmol CYP，4-硝基苯酚2-羟基化分别为139 pmol/min/pmol CYP和277 nl/min/pmol CYP，monCYP2E1的动力学参数表现出相似的值。这些发现表明，人和食蟹猴的CYP2E1酶在氨基酸序列上具有高度同源性，且它们的酶学性质相当相似。本研究获得的信息应有助于体内外推及评估外源化合物的毒性。
