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KMID : 1034820130090010009
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Molecular & Cellular Toxicology
2013 Volume.9 No. 1 p.9 ~ p.14
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Analysis of Substrate Recognition Site 2 (SRS2) in human cytochrome P450 1A2 using whole-plasmid random mutagenesis
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Kim Joo-Hwan
Han Song-Hee
Choi Seung-Hye
Lim Young-Ran
Lee Hwa-Youn
Kim Dong-Hak
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Abstract
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The metabolic activation of many carcinogenic heterocyclic and aryl amines are attributed to hepatic cytochrome P450 enzymes including P450 1A2. Bioactivation mechanism analysis of P450 1A2 in the limited crystallographic data requires the generation of a large number of mutants. In this study, the SRS2 region in P450 1A2 was randomly mutated with random primers designed for the non-wild type using whole-plasmid random mutagenesis, followed the screening of the mutant library. Eight mutants in Asn222 and Glu225 were selected after Escherichia coli genotoxicity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). Each mutant E. coli membrane was used to determine k cat and K m values for phenacetin and 7-ethoxyresorufin O-deethylation. Most mutants showed considerably increased activity for phenacetin. The Glu225Asn and Asn222Lys mutants increased catalytic efficiencies (k cat/K m) by 5- and 4-fold, respectively. However, the catalytic efficiencies of 7-ethoxyresorufin O-deethylation were not increased mainly because of the increase of K m values. The Glu225Asn and Asn222Lys mutants showed increased k cat values but exhibited greater increases in K m values, which resulted in lower catalytic efficiencies (k cat/K m) for 7-ethoxyresorufin O-deethylation. These results suggested that structural changes of metabolic enzymes in the process of mutagenesis result in different effects of activity by its substrates.
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KEYWORD
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P450 1A2, Random mutagenesis, Phenacetin, 7-ethoxyresorufin
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