Oxidation of N-Nitrosoalkylamines by human cytochrome P450 2A6: sequential oxidation to aldehydes and carboxylic acids and analysis of reaction steps.

Chowdhury G, Calcutt MW, Guengerich FP
J Biol Chem. 2010 285 (11): 8031-44

PMID: 20061389 · PMCID: PMC2832954 · DOI:10.1074/jbc.M109.088039

Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH(3)CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect ((D)k(app) approximately 10), which was highly expressed in a variety of competitive and non-competitive experiments. The (D)k(app) for DEN was approximately 3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO(2)H and CH(3)CO(2)H, respectively. In neither case was a lag observed, which was unexpected considering the k(cat) and K(m) parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde).

MeSH Terms (17)

Aldehydes Aryl Hydrocarbon Hydroxylases Binding, Competitive Carbon Radioisotopes Carboxylic Acids Cytochrome P-450 CYP2A6 Deuterium Diethylnitrosamine Dimethylnitrosamine Humans Kinetics Mass Spectrometry Methane Models, Chemical NADP Oxidation-Reduction Substrate Specificity

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