Oxidation of Methoxyphenethylamines by Cytochrome P450 2D6

Abstract

Cytochrome P450 (P450) 2D6 is involved in the oxidation of a large fraction (ϳ30%) of drugs used by humans and also catalyzes the O-demethylation of the model substrates 3-and 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 3-or 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 k cat and k cat /K m 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 2؉ O 2 ؊ 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 k cat , and the contributions of several steps must be considered in understanding rates of the P450 2D6 reactions.

Response to Comment on “Human White Blood Cells Synthesize Morphine: CYP2D6 Modulation”

“…6) In conclusion, our results demonstrating a relatively slow conversion of tyramine, via dopamine, into morphine are supported by our enzymology studies of CYP2D6 showing a 10-fold lower binding affinity of tyramine to the active site of the enzyme as compared with the 3-OH-analog (8,9 …”

“…Additionally, the authors demonstrate conversion of radiolabeled codeine to morphine in WBC, although they have not used appropriate CYP2D6 inhibitors. This is not surprising based on in vitro studies demonstrating that CYP2D6 demethylation occurs faster than ring hydroxylation, i.e., rapid conversion of codeine to morphine (8,9). In light of the above, we do not understand why the authors conclude that WBC do not synthesize morphine.…”

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

“…Deuterated 7-OMe coumarins were prepared by reaction of 7-OH coumarin with deuterated methyl iodides (Cambridge Isotopes, Cambridge, MA) in the usual manner (29,37,38) The synthesis of 3-OH coumarins was done using the general procedure of Neubauer and Flatow (42), which involves condensation of salicylaldehyde or a 4-substituted salicylaldehyde with hippuric acid (N-benzoylglycine) to form the N-benzoyl enamine, which was hydrolyzed in 10 N NaOH (100°C, 45 min) to give the desired coumarin. Enzymes-P450 2A6 was expressed from a plasmid (originally obtained from P. Soucek, National Institute of Public Health, Prague) in Escherichia coli, except that a His 5 tag was attached to the C terminus (24,46).…”

“…Moreover, the observed kinetic isotope effects can very considerably with different P450s and different reactions even though all are of low efficiency (31,34). The variability of observed kinetic isotope effects in various P450 reactions (28,29,31,34,94) is further extended in further work with human P450s 2E1 and 3A4 (95,96). Thus, the branched nature of the P450 reactions can contribute to the higher expression of kinetic isotope effects (92, 93) but is not a sufficient explanation in the absence of further kinetic details about particular reactions.…”

Why Is Quinidine an Inhibitor of Cytochrome P450 2D6?

“…Quinidine is not metabolized by CYP2D6 and has long been established as a potent competitive inhibitor of the enzyme (5)(6)(7)(8)(9). The fact that quinidine is an inhibitor rather than a substrate is intriguing because it produces a classical type I binding spectrum with CYP2D6 (10) that is usually associated with the binding of substrate molecules (11).…”