Felicia N D, Rekha G K, Murphy S E
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Cancer Center, Minneapolis 55455, USA.
Arch Biochem Biophys. 2000 Dec 15;384(2):418-24. doi: 10.1006/abbi.2000.2128.
The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen in the A/J mouse, and is believed to be a causative agent for human lung cancer. NNK requires metabolic activation by alpha-hydroxylation to exert its carcinogenic potential. The human P450, 2A6 is a catalyst of this reaction. There are two closely related enzymes in the mouse, P450 2A4 and 2A5, which differ from each other by only 11 amino acids. In the present study these two mouse P450s were expressed in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. The catalysis of NNK metabolism by Sf9 microsomal fractions containing either P450 2A4 or 2A5 was determined. Both enzymes catalyzed the alpha-hydroxylation of NNK but with strikingly different efficiencies and specificities. P450 2A5 preferentially catalyzed NNK methyl hydroxylation, while P450 2A4 preferentially catalyzed methylene hydroxylation. The KM and Vmax for the former were 1.5 microM and 4.0 nmol/min/nmol P450, respectively, and for the latter 3.9 mM and 190 nmol/min/nmol P450. The mouse coumarin 7-hydroxylase, P450 2A5 is a significantly better catalyst of NNK alpha-hydroxylation than is the closely related human enzyme, P450 2A6.
烟草特异性亚硝胺4-(甲基亚硝氨基)-1-(3-吡啶基)-1-丁酮(NNK)是A/J小鼠中一种强效的肺癌致癌物，被认为是人类肺癌的致病因子。NNK需要通过α-羟基化进行代谢激活才能发挥其致癌潜力。人类细胞色素P450 2A6是该反应的催化剂。小鼠中有两种密切相关的酶，P450 2A4和2A5，它们彼此仅相差11个氨基酸。在本研究中，这两种小鼠P450酶利用重组杆状病毒在草地贪夜蛾(Sf9)细胞中表达。测定了含有P450 2A4或2A5的Sf9微粒体组分对NNK代谢的催化作用。两种酶都催化NNK的α-羟基化，但效率和特异性明显不同。P450 2A5优先催化NNK甲基羟基化，而P450 2A4优先催化亚甲基羟基化。前者的米氏常数(KM)和最大反应速度(Vmax)分别为1.5微摩尔/升和4.0纳摩尔/分钟/纳摩尔P450，后者分别为3.9毫摩尔/升和190纳摩尔/分钟/纳摩尔P450。小鼠香豆素7-羟化酶P450 2A5对NNKα-羟基化的催化作用明显优于密切相关的人类酶P450 2A6。
