Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450

Liver microsomal cytochrome P-450 readily N-dealkylates N,N-dimethylamides. N-Methyl-N-hydroxymethyl amides were isolated as intermediates and characterized by gas chromatography-mass spectrometry as their trimethylsilyl ethers. Intramolecular kinetic deuterium isotope effects measured for the enzymic N-demethylation of a series of 12 aromatic and aliphatic N-methyl-N-trideuteriomethyl amides, RCON(CH3)CD3, varied from 3.6 to 6.9 but were independent of both amide bond rotation rate and substrate oxidation potential. These values, which represent a lower limit to the intrinsic isotope effect (Dkintrinsic), are significantly larger than those observed for anodic N-demethylation and are consistent with a mechanism involving hydrogen atom abstraction. On the other hand, with N,N-dimethylbenzamide the intermolecular kinetic deuterium isotope effects on Vmax and Vmax/Km were found to be much smaller (1.23 and 1.75, respectively) indicating substantial suppression of the intrinsic isotope effect. Such suppression indicates the occurrence of a rate-limiting step other than the isotopically sensitive step together with a strong commitment to catalysis.