TY - JOUR T1 - Functional Characterization of Cytochrome P450 2A6 Allelic Variants <em>CYP2A6*15</em>, <em>CYP2A6*16</em>, <em>CYP2A6*21</em>, and <em>CYP2A6*22</em> JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 745 LP - 751 DO - 10.1124/dmd.109.031054 VL - 38 IS - 5 AU - Kai Hung Tiong AU - Beow Chin Yiap AU - Eng Lai Tan AU - Rusli Ismail AU - Chin Eng Ong Y1 - 2010/05/01 UR - http://dmd.aspetjournals.org/content/38/5/745.abstract N2 - 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 CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22, 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 CYP2A6*15, R203S substitution in CYP2A6*16, K476R substitution in CYP2A6*21, and concurrent D158E and L160I substitutions in CYP2A6*22. 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 CYP2A6*15 exhibited higher Vmax than the wild type, whereas all mutant constructs, except for variant CYP2A6*16, exhibited higher Km values. Analysis of the Vmax/Km ratio revealed that all mutants demonstrated 0.85- to 1.05-fold differences from the wild type, with the exception of variant CYP2A6*22, which only portrayed 39% of the wild-type intrinsic clearance. These data suggested that individuals carrying the CYP2A6*22 allele are likely to have lower metabolism of CYP2A6 substrate than individuals expressing CYP2A6*15, CYP2A6*16, CYP2A6*21, and the wild type. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics ER -