Oxidation of caffeine by CYP1A2: isotope effects and metabolic switching

Caffeine (1,3,7-trimethylxanthine) has previously been shown to undergo metabolic switching in vivo when the N-1 or the N-7 methyl groups were trideuteromethylated [Horning et al. (1976) Proceedings of the Second International Conference on Stable Isotopes, pp 41-54]. We have examined the effect of replacing the N-3 methyl group with a trideuteromethyl group. The corresponding isotope effects can then be used to distinguish the kinetic mechanism by which four primary metabolites can be formed from one substrate by one cytochrome P450 (P450). We have synthesized 3-CD3-caffeine and 3-CD3-7-CD3-caffeine as well as trideuteromethylated analogs of each of the in vitro metabolites formed by cytochrome P4501A2. The observed competitive isotope effects for the metabolites, which do not result from deuterium abstraction (theobromine, theophylline), demonstrate that the nondissociative mechanism applies to caffeine metabolism by cytochrome P4501A2. Thus, there must be equilibration of the kinetically distinguishable activated P450-substrate complexes at rates competitive with hydrogen abstraction. The true isotope effects for the N-3 demethylation of caffeine were derived from the ratios of the amount of paraxanthine relative to the amount of theobromine or theophylline. The resultant ratios indicate that these isotope effects are essentially intrinsic. Observation of the isotope effects on N-3 demethylation was facilitated by branching to the minor in vitro metabolites as well as water formation. Product release is not rate-limiting for this system.