Zhou Huijia, Josephy P David, Kim Donghak, Guengerich F Peter
Department of Chemistry and Biochemistry, Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Guelph, Ont. N1G 2W1, Guelph, Canada.
Arch Biochem Biophys. 2004 Feb 1;422(1):23-30. doi: 10.1016/j.abb.2003.11.019.
Human cytochrome P450 1A2 catalyzes important reactions in xenobiotic metabolism, including the N-hydroxylation of carcinogenic aromatic amines. In 2001, Chevalier et al. reported four new P450 1A2 sequence variants in the human population. We have now expressed these variants in Escherichia coli and measured protein expression (optical spectroscopy of holoenzyme and immunoblotting) and bioactivation of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and MeIQ (2-amino-2,4-dimethylimidazo[4,5-f]quinoline) in the lacZ reversion mutagenicity test. Enzyme kinetic analyses were performed for N-hydroxylation of five heterocyclic amine substrates and for O-deethylation of phenacetin. The most drastic effect was that of the R431W substitution: no holoenzyme was detectable. This residue is located in the "meander" peptide region and earlier site-directed mutagenesis studies demonstrated that it is critical for maintenance of protein tertiary structure. The other three variants had subtly different catalytic activities compared to the wild-type enzyme.
人类细胞色素P450 1A2催化外源性物质代谢中的重要反应，包括致癌芳香胺的N-羟基化反应。2001年，谢瓦利埃等人报告了人类群体中四个新的P450 1A2序列变体。我们现在已在大肠杆菌中表达了这些变体，并在lacZ回复突变试验中测量了蛋白质表达（全酶的光谱学和免疫印迹）以及IQ（2-氨基-3-甲基咪唑并[4,5-f]喹啉）和MeIQ（2-氨基-2,4-二甲基咪唑并[4,5-f]喹啉）的生物活化作用。对五种杂环胺底物的N-羟基化反应以及非那西丁的O-脱乙基反应进行了酶动力学分析。影响最为显著的是R431W替代：未检测到全酶。该残基位于“曲折”肽区域，早期的定点诱变研究表明它对维持蛋白质三级结构至关重要。与野生型酶相比，其他三个变体具有略有不同的催化活性。
