Kinetic deuterium isotope effects on deamination and N-hydroxylation of cyclohexylamine by rabbit liver microsomes

Abstract

Deuterium isotope effects on the kinetic parameters for deamination and N-hydroxylation of cyclohexylamine (CHA) catalyzed by rabbit liver microsomes with NADPH are investigated. Both reactions are inhibited by carbon monoxide and have the characteristics of typical cytochrome P450-dependent monooxygenase reactions. A small and significant deuterium isotope effect operates in the oxidative deamination of CHA. The apparent isotope effects i.e., V H V D and ( V K) H ( V K ratios for deamination, are 1.75 and 1.8–2.3, respectively. On the basis of N-hydroxylation, the V H V D and V K ratios are 0.8–0.9. The N-hydroxylation rate of α-deuterated CHA (D-CHA) is somewhat higher than that of CHA. The increased increment of hydroxylamine formation seems to coincide with the decreased amount of deamination. Substitution of deuterium in the α-position of CHA results in metabolic switching of cytochrome P450 from deamination to N-hydroxylation with low deuterium isotope effects. The data are interpreted in terms of an initial one-electron abstraction from the nitrogen to form an aminium cation radical followed by recombination with iron-bound hydroxyl radical leading to N-hydroxylamine, or followed by α-carbon deprotonation to form a neutral carbon radical. The latter can lead to a carbinolamine intermediate for deamination by way of imine or recombination with nascent iron-bound hydroxyl radical. The relative rates of the reactions depend on the α-carbon deprotonation rates of amines.