Oxidation of Methoxyphenethylamines by Cytochrome P450 2D6

Cytochrome P450 (P450) 2D6 is involved in the oxidation of a large fraction ( 30%) of drugs used by humans and also catalyzes the O-demethylation of the model substrates 3and 4-methoxyphenethylamine followed by subsequent ring hydroxylation to dopamine. Burst kinetics were not observed; rate-limiting step(s) must occur prior to product formation. Rates of reduction of ferric P450 2D6 were stimulated by 3or 4-methoxyphenethylamine or the inhibitor quinidine; reduction is not the most rate-limiting step. The non-competitive intramolecular deuterium isotope effect, an estimate of the intrinsic isotope effect, for 4-methoxyphenethylamine O-demethylation was 9.6. Intermolecular noncompetitive deuterium isotope effects of 3.1–3.8 were measured for kcat and kcat/Km for both O-demethylation reactions, implicating at least partially rate-limiting C–H bond breaking. Simulation of steady-state kinetic data yielded a catalytic mechanism dominated by the rates of (i) Fe O2 protonation (plus O–O bond scission) and (ii) C–H bond breaking, consistent with the appearance of the spectral intermediates in the steady state, attributed to iron–oxygen complexes. However, all the rates of individual steps (or rates of combined steps) are considerably higher than kcat, and the contributions of several steps must be considered in understanding rates of the P450 2D6 reactions.