Analysis of Coumarin 7-Hydroxylation Activity of Cytochrome P450 2A6 using Random Mutagenesis

摘要: Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination random mutagenesis high-throughput screening was used analysis 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (kcat Km) for by wild-type 35 selected mutants were measured indicated that throughout coding region can have effects on activity. Five showing decreased catalytic efficiency (kcat/Km) further analyzed substrate selectivity binding affinities showed reduced activities 7-methoxycoumarin O-demethylation, tert-butyl methyl ether indole 3-hydroxylation. All except one (K476E) (and also higher Km values), indicating this major basis enzymatic activities. recent x-ray crystal structure bound to (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., Johnson, E. F. (2005) Nat. Struct. Mol. Biol. 12, 822-823) indicates recovered A481T N297S mutations appear be close suggesting direct perturbation interaction. activity K476E mutant associated with decreases both NADPH oxidation reduction rate ferric 2A6-coumarin complex. attenuation caused part lower affinity NADPH-P450 reductase, but did not achieve even at high reductase concentrations.