Oxidation of Methyl and Ethyl Nitrosamines by Cytochromes P450 2E1 and 2B1

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 non-competitive 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 non-competitive 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 for ethyl compared to 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, lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkylnitrosamine oxidation appears to be shared by a number of P450s.