Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism

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• 作者：Keizers ; Peter H.J. ; Lussenburg ; Barbara M.A. ; de Graaf ; Chris ; Mentink ; Letty M. ; Vermeulen ; Nico P.E. ; et. al.
• 关键词：CYP ; cytochrome P450 ; MAMC ; 7-methoxy-4-(aminomethyl)-coumarin ; Phe¹²⁰Ala ; phenylalanine 120 &rarr ; alanine ; MDMA ; 3 ; 4-methylenedioxymethylamphetamine ; 3 ; 4-OH-MA ; 3 ; 4-dihydroxymethamphetamine ; MDA ; 3 ; 4-methylenedioxyamphetamine ; N-OH-MDM
• 刊名：Biochemical Pharmacology
• 出版年：2004
• 出版时间：December 1, 2004
• 年：2004
• 卷：68
• 期：11
• 页码：2263-2271
• 全文大小：253 K

文摘

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 (Phe¹²⁰). In this study, the role of Phe¹²⁰ 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: 1.2μM, wild-type: 2.9μM, 4-hydroxylation mutant: 1.5μM, and wild-type: 3.2μM) and neither was affected dextromethorphan O-demethylation (K_m mutant: 1.2μM, wild-type: 2μM, k_cat mutant: 4.5min⁻¹, and wild-type: 3.3min⁻¹). However, the Phe¹²⁰Ala 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: 55μM and wild-type: 2μM). 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 Phe¹²⁰Ala mutant for quinidine (IC₅₀ mutant: 240nM and wild-type, 40nM), while IC₅₀s for quinine were equal (1μM). These data indicate the importance of Phe¹²⁰ in the selectivity and regiospecificity in substrate binding and catalysis by CYP2D6.