Identification of critical amino acid residues of human CYP2A13 for the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific carcinogen

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 (Ala(117)Val, His(164)Gly, Ser(208)Ile, His(372)Arg, and Pro(465)Ser). 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 alpha-hydroxylation. We demonstrated that, except for the His(164)Gly mutant, all the CYP2A13 mutant proteins showed a significant decrease in the catalytic efficiency (V-max/K-m) for NNK alpha-hydroxylation. The His(372) 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 Ala(117) 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 Ala(117)Val and His(372)Arg 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 information of human CYP2A13.