Abstract

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 human. The present study aimed to investigate the reported allele CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22, in terms of the functional consequences of their mutations on the enzyme catalytic activity. Utilizing the wild type CYP2A6 cDNA as template, site-directed mutagenesis was carried out to introduce nucleotide changes encoding Lys194Glu substitution in CYP2A6*15, Arg203Ser substitution in CYP2A6*16, Lys476Arg substitution in CYP2A6*21 and concurrent Asp158Glu and Leu160Ile substitutions in CYP2A6*22. Upon sequence verification, the CYP2A6 wild type and mutant constructs were individually co-expressed with NADPH CYP-reductase in E. coli. Kinetic study using coumarin 7-hydroxylase assay indicated that CYP2A6*15 exhibited higer V_max than the wild type while all mutant constructs, except for variant CYP2A6*16, exhibited higher K_m values. Analysis of V_max/K_m ratio revealed that all mutants demonstrated 0.85- to 1.05-fold difference from the wild type, with exception of variant CYP2A6*22 which only portrayed 39% of the wild-type intrinsic clearance. These data suggested that individuals carrying 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.