title: Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.
abstract: Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine [N,N-dimethylnitrosamine (DMN)], a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥8. The KIE was not attenuated in noncompetitive intermolecular experiments with rat liver microsomes {(D)V = 12.5; (D)(V/K) = 10.9 [nomenclature of Northrop, D. B. (1982) Methods Enzymol. 87, 607-625]} but was with purified human P450 2E1 [(D)V = 3.3; (D)(V/K) = 3.7], indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine [N,N-diethylnitrosamine (DEN)], the intrinsic KIE was slightly lower and was not expressed [e.g., (D)(V/K) = 1.2] in noncompetitive intermolecular experiments. The same general pattern of KIEs was also seen in the (D)(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH(3)NH(2), CH(3)CH(2)NH(2), and NO(2)(-)). Experiments with deuterated N-nitroso-N-methyl-N-ethylamine demonstrated that the lower KIEs associated with ethyl versus methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 [Chowdhury, G., et al. (2010) J. Biol. Chem. 285, 8031-8044]. These same features (no lag phase for HCO(2)H formation and a lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has a lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkyl nitrosamine oxidation appears to be shared by a number of P450s.
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
authors.forename: Goutam
authors.initials: G
authors.lastname: Chowdhury
authors.forename: M Wade
authors.initials: MW
authors.lastname: Calcutt
authors.forename: Leslie D
authors.initials: LD
authors.lastname: Nagy
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D000818:Animals; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism*; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism; D004052:Diethylnitrosamine / Q000378:metabolism*; D004128:Dimethylnitrosamine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D004052:Diethylnitrosamine; D003903:Deuterium; D019392:Cytochrome P-450 CYP2E1; D019362:Cytochrome P-450 CYP2B1; D004128:Dimethylnitrosamine