Characterization of cytochrome P450 2A4 and 2A5-catalyzed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-but anone (NNK), is a potent lung carcinogen in the A/J mouse, and is believed to be a causative agent for human lung cancer. NNK requires metabolic activ ation by alpha -hydroxylation to exert its carcinogenic potential. The huma n P450, 2A6 is a catalyst of this reaction. There are two closely related e nzymes in the mouse, P450 2A4 and 2A5, which differ from each other by only II amino acids. In the present study these two mouse P450s were expressed in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. The cat alysis of NNK metabolism by Sf9 microsomal fractions containing either P450 2A4 or 2A5 was determined. Both enzymes catalyzed the alpha -hydroxylation of NNK but with strikingly different efficiencies and specificities. P450 2A5 preferentially catalyzed NNK methylhydroxylation, while P450 2A4 prefer entially catalyzed methylene hydroxylation. The K-M and V-max for the forme r were 1.5 muM and 4.0 nmol/min/nmol P450, respectively, and for the latter 3.9 mM and 190 nmol/min/nmol P450. The mouse coumarin 7-hydroxylase, P450 2A5 is a significantly better catalyst of NNK alpha -hydroxylation than is the closely related human enzyme, P450 2A6. (C) 2000 Academic Press.