Analysis of Substrate Recognition Site 2 (SRS2) in human cytochrome P450 1A2 using whole-plasmid random mutagenesis

摘要: The metabolic activation of many carcinogenic heterocyclic and aryl amines are attributed to hepatic cytochrome P450 enzymes including 1A2. Bioactivation mechanism analysis 1A2 in the limited crystallographic data requires generation a large number mutants. In this study, SRS2 region was randomly mutated with random primers designed for non-wild type using whole-plasmid mutagenesis, followed screening mutant library. Eight mutants Asn222 Glu225 were selected after Escherichia coli genotoxicity assay involving reversion lac prototrophy as response amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). Each E. membrane used determine k cat K m values phenacetin 7-ethoxyresorufin O-deethylation. Most showed considerably increased activity phenacetin. Glu225Asn Asn222Lys catalytic efficiencies (k cat/K m) by 5- 4-fold, respectively. However, O-deethylation not mainly because increase values. but exhibited greater increases values, which resulted lower These results suggested that structural changes process mutagenesis result different effects its substrates.