Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism.
The polymorphic human debrisoquine hydroxylase, cytochrome P450 2D6 (CYP2D6), 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 modelling studies, it has been suggested that in fixation of this aromatic moiety, there may be an important role for phenylalanine 120 (Phe(120)). In this study, the role of Phe(120) 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 CYP2D6. On the other hand, bufuralol metabolism was hardly affected (K(m) of 1-hydroxylation mutant: 1.2 microM, wild-type: 2.9 microM, 4-hydroxylation mutant: 1.5 microM, and wild-type: 3.2 microM) and neither was affected dextromethorphan O-demethylation (K(m) mutant: 1.2 microM, wild-type: 2 microM, k(cat) mutant: 4.5 min(-1), and wild-type: 3.3 min(-1)). However, the Phe(120)Ala mutant also formed 3-hydroxymorphinan, the double demethylated form of dextromethorphan, which was not detected using wild-type CYP2D6. 3,4-Methylenedioxymethamphetamine (MDMA) was demethylenated by both mutant and wild-type CYP2D6 to 3,4-dihydroxymethamphetamine (3,4-OH-MA K(m) of mutant: 55 microM and wild-type: 2 microM). In addition, the mutant formed two additional metabolites; 3,4-methylenedioxyamphetamine (MDA) and N-hydroxy-3,4-methylenedioxymethamphetamine (N-OH-MDMA). Inhibition experiments of dextromethorphan O-demethylation showed a decreased affinity of the Phe(120)Ala mutant for quinidine (IC(50) mutant: 240 nM and wild-type, 40 nM), while IC(50)s for quinine were equal (1 microM). These data indicate the importance of Phe(120) in the selectivity and regiospecificity in substrate binding and catalysis by CYP2D6.
Keizers
Peter H J
PH
LACDR/Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Lussenburg
Barbara M A
BM
de Graaf
Chris
C
Mentink
Letty M
LM
Vermeulen
Nico P E
NP
Commandeur
Jan N M
JN
D000409:Alanine / Q000235:genetics; D003374:Coumarins / Q000378:metabolism*; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism*; D003915:Dextromethorphan / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D016296:Mutagenesis; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*
C120605:7-methoxy-4-(aminomethyl)coumarin; D003374:Coumarins; D010649:Phenylalanine; D003915:Dextromethorphan; D019389:Cytochrome P-450 CYP2D6; D018817:N-Methyl-3,4-methylenedioxyamphetamine; D000409:Alanine