USING SITE-DIRECTED MUTAGENESIS FOR DETERMINATION OF SUBSTRATE INTERACTION RESIDUES IN CYTOCHROME P450 2D6 part I

The polymorphic human debrisoquine hydroxylase, Cytochrome P450 2D6 (P450 2D6), is one of the most important phase I drug metabolising enzymes. It is responsible for metabolising a large number of compounds that mostly share similarity in having a basic N-atom and an aromatic moiety. In homology modeling studies it has been suggested that in fixation of this aromatic moiety there may be an important role for phenylalanine 120 (F120). In this study the role of F120 in ligand binding and catalysis was experimentally examined by mutating it into an alanine. Strikingly, this substitution led to a completely abolished 7-methoxy-4(aminomethyl)-coumarin (MAMC) O-demethylating activity of P450 2D6. On the other hand, bufuralol metabolism was hardly affected (Km 1-hydroxylation mutant: 1.2 μM, wild-type: 2.9 μM, 4-hydroxylation mutant: 1.5 μM, wild-type: 3.2 μM) and neither was dextromethorphan O-demethylation (Km mutant: 1.2 μM, wild-type: 2 μM, Vmax mutant: 4.5 min-1, wild-type: 3.3 min-1). However, the F120A mutant also formed 3-hydroxymorphinan, the double demethylated form of dextromethorphan, which was not detected using wild-type P450 2D6. 3,4-methylenedioxymethylamphetamine (MDMA) was demethylenated by both mutant and wild-type P450 2D6 to 3,4-dihydroxymethamphetamine (3,4-OH-MA, Km mutant: 55 μM, wild-type: 2 μM). In addition the mutant formed two additional metabolites; 3,4-methylenedioxyamphetamine (MDA) and N-hydroxy-3,4-methylenedioxymethylamphetamine (N-OH-MDMA). Inhibition experiments of dextromethorphan O-demethylation showed a decreased affinity of the F120A mutant for quinidine (IC50 mutant: 240 nM, wild-type: 40 nM), while IC50’s for quinine were equal (1 μM). These data indicate the importance of F120 in the selectivity and regiospecificity in substrate binding and catalysis by P450 2D6.