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Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.
Chowdhury, Goutam; Calcutt, M Wade; Nagy, Leslie D; Guengerich, F Peter.
Afiliación
  • Chowdhury G; Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
Biochemistry ; 51(50): 9995-10007, 2012 Dec 18.
Article en En | MEDLINE | ID: mdl-23186213
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.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Citocromo P-450 CYP2B1 / Citocromo P-450 CYP2E1 / Dietilnitrosamina / Dimetilnitrosamina Límite: Animals / Humans / Male Idioma: En Revista: Biochemistry Año: 2012 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Citocromo P-450 CYP2B1 / Citocromo P-450 CYP2E1 / Dietilnitrosamina / Dimetilnitrosamina Límite: Animals / Humans / Male Idioma: En Revista: Biochemistry Año: 2012 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos