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 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.
Footnotes
This work was supported by the Malaysia Toray Science Foundation [Science and Technology Research Grant 269817-K]; and by the International Medical University, Kuala Lumpur, Malaysia [International Medical University Research Fund IMU 112/2006].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.031054.
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ABBREVIATIONS:
- P450
- cytochrome P450
- OxR
- NADPH-cytochrome P450 oxidoreductase
- SRS
- substrate recognition site.
- Received November 13, 2009.
- Accepted February 5, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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