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Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism.
Biochem Pharmacol. 2004 Dec 01; 68(11):2263-71.BP

Abstract

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.

Authors+Show Affiliations

LACDR/Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15498516

Citation

Keizers, Peter H J., et al. "Influence of Phenylalanine 120 On Cytochrome P450 2D6 Catalytic Selectivity and Regiospecificity: Crucial Role in 7-methoxy-4-(aminomethyl)-coumarin Metabolism." Biochemical Pharmacology, vol. 68, no. 11, 2004, pp. 2263-71.
Keizers PH, Lussenburg BM, de Graaf C, et al. Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism. Biochem Pharmacol. 2004;68(11):2263-71.
Keizers, P. H., Lussenburg, B. M., de Graaf, C., Mentink, L. M., Vermeulen, N. P., & Commandeur, J. N. (2004). Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism. Biochemical Pharmacology, 68(11), 2263-71.
Keizers PH, et al. Influence of Phenylalanine 120 On Cytochrome P450 2D6 Catalytic Selectivity and Regiospecificity: Crucial Role in 7-methoxy-4-(aminomethyl)-coumarin Metabolism. Biochem Pharmacol. 2004 Dec 1;68(11):2263-71. PubMed PMID: 15498516.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism. AU - Keizers,Peter H J, AU - Lussenburg,Barbara M A, AU - de Graaf,Chris, AU - Mentink,Letty M, AU - Vermeulen,Nico P E, AU - Commandeur,Jan N M, PY - 2004/05/17/received PY - 2004/08/02/accepted PY - 2004/10/23/pubmed PY - 2005/3/1/medline PY - 2004/10/23/entrez SP - 2263 EP - 71 JF - Biochemical pharmacology JO - Biochem Pharmacol VL - 68 IS - 11 N2 - 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. SN - 0006-2952 UR - https://www.unboundmedicine.com/medline/citation/15498516/Influence_of_phenylalanine_120_on_cytochrome_P450_2D6_catalytic_selectivity_and_regiospecificity:_crucial_role_in_7_methoxy_4__aminomethyl__coumarin_metabolism_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0006-2952(04)00593-3 DB - PRIME DP - Unbound Medicine ER -