RT Journal Article
SR Electronic
T1 Functional characterization of cytochrome P450 2A6 (CYP2A6) allelic variants CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP dmd.109.031054
DO 10.1124/dmd.109.031054
A1 Tiong, Kai Hung
A1 Yap, Beow Chin
A1 Tan, Eng Lai
A1 Ismail, Rusli
A1 Ong, Chin Eng
YR 2010
UL http://dmd.aspetjournals.org/content/early/2010/02/05/dmd.109.031054.abstract
AB 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 Vmax than the wild type while all mutant constructs, except for variant CYP2A6*16, exhibited higher Km values. Analysis of Vmax/Km 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.The American Society for Pharmacology and Experimental Therapeutics