Elsevier

Biochemical Pharmacology

Volume 68, Issue 11, 1 December 2004, Pages 2263-2271
Biochemical Pharmacology

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

https://doi.org/10.1016/j.bcp.2004.08.013Get rights and content

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

Introduction

Cytochromes P450 (CYPs) are heme-containing enzymes capable of oxidizing and reducing a large variety of endogenous and exogenous substrates in virtually all living organisms [1], [2]. In humans, one of the most important hepatic phase I drug metabolising enzymes is CYP2D6. It is involved in the metabolism of about 30% of the currently marketed drugs, including neuroleptics, antidepressants β-blockers, opioids and antiarythmics [3], [4]. The enzyme is known for its genetic polymorphisms, even increasing its clinical relevance [5], [6]. Although some crystal structures of mammalian CYPs have become available in recent years [7], [8], so far no crystal structure of CYP2D6 has been resolved. Structural information on this enzyme still depends on homology modelling and mutagenesis studies.

Recently we have developed a new homology model of CYP2D6 based on the crystal structure of rabbit CYP2C5 [9]. This was the first model of CYP2D6 based on a mammalian CYP template and is therefore considered an improvement over the existing models. This was indicated by the good correlation between experimental data and the modelled protein–substrate interactions. The model identified some active site key residues. First of all, Glu216 has been shown to be a key ligand-binding residue involved in hydrogen bonding with a variety of substrates. This residue has been subjected to several mutagenesis studies [10], [11] that indicated involvement of this residue in fixation of basic nitrogen atoms present in many CYP2D6 substrates. A second important active site residue in this and in other homology models is Phe483[12], interacting via van der Waals forces with substrates like codeine. Earlier mutagenesis of this residue to an isoleucine showed effects on testosterone metabolism [13]. However, the anchoring of aromatic moieties present in most CYP2D6 substrates, as predicted in pharmacophore models [14], [15], [16], cannot be completely ascribed to this residue. Therefore, other Phe residues contributing to aromatic interactions with substrates have to be present in the active site. Mutation of Phe481 to other non-aromatic residues led to decreased activities towards model substrates [17], although in the model of Venhorst et al., this residue is not considered to be in the active site of CYP2D6.

Even more interesting is the position of a third phenylalanine at position 120 in the active site of CYP2D6 homology models [9], [11], [12]. In these CYP2C5-based models Phe120 appears to be positioned directly above the porphyrin ring. From docking and molecular dynamics studies, this residue seems to be the anchoring residue for the aromatic moiety of ligands like quinidine and sparteine via π–π stacking [9]. In the present study, the role of Phe120 in ligand binding and metabolism by CYP2D6 is studied experimentally by mutating this residue into an alanine. According to computational simulations, this mutation will not only create more space in the active site, but also eliminate a potentially important aromatic anchoring point. By mutating Phe120 into Ala, we are aiming at the elucidation of the role of this residue in substrate binding and turnover by CYP2D6.

Section snippets

Materials

The pSP19T7LT_2D6 plasmid containing human 2D6 with a C-terminal His6-tag bicistronically co-expressed with human cytochrome P450 NADPH reductase was kindly provided by Prof. Dr. Ingelman-Sundberg. 7-Methoxy-4-(aminomethyl)-coumarin (MAMC), 7-hydroxy-4-(aminomethyl)-coumarin (HAMC), 3,4-methylenedioxymethylamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) were synthesised as described [18], [19]. Bufuralol hydrochloride was obtained from Gentest. N-Methylhydroxylamine hydrochloride,

Expression of wild-type and Phe120Ala mutant CYP2D6

The Phe120Ala mutant had consistently significantly lower expression levels compared to wild-type CYP2D6. An average culture contained about 70 nM of CYP for the mutant versus 200 nM for wild-type after 48 h induction. In the difference spectrum taken from the culture of the mutant enzyme, a high absorbance was measured at 420 nm showing the presence of large amounts of P420, the inactive form of CYP, indicating that the mutation decreases the stability of the enzyme to some extent.

Metabolism of model compounds

O-Demethylation

Discussion

Recently computer homology modelling studies suggested Phe120 to be a CYP2D6 active site residue involved in the binding of substrates via aromatic interactions [9], [11], [12]. The primary aim of this study was to evaluate the role of this residue in the CYP2D6 active site. The results presented in this study show that Phe120 is very relevant in CYP2D6 ligand binding, substrate selectivity and regiospecificity in catalysis.

The expression levels of the Phe120Ala mutant in E. coli were found to

Acknowledgements

We would like to thank Dr. Frans de Kanter for his help with recording and elucidating the NMR spectra and Ed Groot for his help in preparing the E. coli membranes.

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