Abstract

Among all the known human cytochrome P450 enzymes, CYP2A13 has the highest efficiency in catalyzing the metabolic activation (keto aldehyde and keto alcohol formation) of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent lung carcinogen in animals and a suspected human lung carcinogen. As part of the structure-activity relationship (SAR) study, the present work was done to identify the key amino acid residues in CYP2A13 that are responsible for this high catalytic efficiency by using a series of mutants (Ala¹¹⁷Val, His¹⁶⁴Gly, Ser²⁰⁸Ile, His³⁷²Arg, and Pro⁴⁶⁵Ser). In these CYP2A13 mutants, the amino acid residues were substituted by the residues at the corresponding positions of CYP2A6, which shares 93.5% amino acid sequence identity with CYP2A13 but is significantly less active (<5%) than CYP2A13 in NNK α-hydroxylation. We demonstrated that, except for the His¹⁶⁴Gly mutant, all the CYP2A13 mutant proteins showed a significant decrease in the catalytic efficiency (V_max/K_m) for NNK α-hydroxylation. The His³⁷² to Arg substitution resulted in a 20-fold increase in the K_m value and a 7-fold decrease in the V_max value for keto aldehyde formation as well as a total loss of detectable keto alcohol formation. The Ala¹¹⁷ to Val substitution, however, only caused a selective decrease in the V_max value for keto aldehyde formation. The role of these amino acid residues in CYP2A13-catalyzed reactions is clearly substrate-dependent, since the same Ala¹¹⁷Val and His³⁷²Arg mutants showed a 9-fold increase in the catalytic efficiency for coumarin 7-hydroxylation. Together with the computational substrate docking, our study provides new SAR in formation of human CYP2A13.