School of Pharmacy and Health Sciences, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia.
Drug Metab Dispos. 2010 May;38(5):745-51. doi: 10.1124/dmd.109.031054. Epub 2010 Feb 5.
Variation in CYP2A6 levels and activity can be attributed to genetic polymorphism and, thus, functional characterization of allelic variants is necessary to define the importance of CYP2A6 polymorphism in humans. The aim of the present study was to investigate the reported alleles CYP2A615, CYP2A616, CYP2A621, and CYP2A622, in terms of the functional consequences of their mutations on the enzyme catalytic activity. With use of the wild-type CYP2A6 cDNA as template, site-directed mutagenesis was performed to introduce nucleotide changes encoding K194E substitution in CYP2A615, R203S substitution in CYP2A616, K476R substitution in CYP2A621, and concurrent D158E and L160I substitutions in CYP2A622. Upon sequence verification, the CYP2A6 wild-type and mutant constructs were individually coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. A kinetic study using a coumarin 7-hydroxylase assay indicated that CYP2A615 exhibited higher V(max) than the wild type, whereas all mutant constructs, except for variant CYP2A616, exhibited higher K(m) values. Analysis of the V(max)/K(m) ratio revealed that all mutants demonstrated 0.85- to 1.05-fold differences from the wild type, with the exception of variant CYP2A622, which only portrayed 39% of the wild-type intrinsic clearance. These data suggested that individuals carrying the CYP2A622 allele are likely to have lower metabolism of CYP2A6 substrate than individuals expressing CYP2A615, CYP2A616, CYP2A6*21, and the wild type.
CYP2A6 水平和活性的变化可归因于遗传多态性，因此，有必要对等位基因变异体进行功能特征分析，以确定 CYP2A6 多态性在人类中的重要性。本研究旨在研究报告的 CYP2A615、CYP2A616、CYP2A621 和 CYP2A622 等位基因，研究其突变对酶催化活性的功能后果。使用野生型 CYP2A6 cDNA 作为模板，通过定点突变引入编码 CYP2A615 中的 K194E 取代、CYP2A616 中的 R203S 取代、CYP2A621 中的 K476R 取代和 CYP2A622 中的 D158E 和 L160I 取代的核苷酸变化。经过序列验证后，将 CYP2A6 野生型和突变构建体分别与 NADPH-细胞色素 P450 还原酶在大肠杆菌中共同表达。使用香豆素 7-羟化酶测定法进行的动力学研究表明，CYP2A615 表现出比野生型更高的 Vmax，而除了变体 CYP2A616 之外，所有突变构建体均表现出更高的 K m 值。Vmax/Km 比值分析表明，除了变体 CYP2A622 之外，所有突变体与野生型相比均表现出 0.85-1.05 倍的差异，而 CYP2A622 仅表现出野生型内在清除率的 39%。这些数据表明，携带 CYP2A622 等位基因的个体可能比表达 CYP2A615、CYP2A616、CYP2A621 和野生型的个体具有更低的 CYP2A6 底物代谢。
