Homology models of cytochrome P450 2D6 (CYP2D6) have identified serine 304 as an active-site residue and implicated a putative role for this residue in substrate enantioselectivity and the differential inhibition of enzyme activity by the diastereoisomers quinine and quinidine. The role of serine 304 in selectivity is thought to be achieved through a preferential hydrogen-bond interaction between the hydroxyl group of the residue and one of the stereoisomers of each ligand. We have tested this hypothesis by substituting serine 304 with alanine, a non-hydrogen-bonding residue, and compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived enzyme. The Ser304Ala substitution did not alter the enantioselective oxidation of metoprolol; the O-demethylation reaction remained R-(+)-enantioselective (wild-type, RS, 1.7; mutant, RS, 1.6), whereas α-hydroxylation remained S-(-)-enantioselective (wild-type and mutant, R/S, 0.7). Similarly, the selective oxidation of the R-(+) and S-(-) enantiomers of propranolol to the major 4-hydroxy metabolite was identical with both wild-type and mutant forms of the enzyme (R/S 0.9), although the formation of minor metabolites (5-hydroxy and deisopropylpropranolol) did show some slight alteration in enantioselectivity. The differential inhibition of enzyme activity by quinine and quinidine was also identical with both forms of CYP2D6, the IC50 values for each enzyme being approx. 10 μM and 0.1 μM for quinine and quinidine, respectively. The kinetics of formation of α-hydroxymetoprolol and 4-hydroxydebrisoquine by wild-type and the Ser304Ala mutant was also very similar. However, modest changes in the regioselective oxidation of metoprolol and debrisoquine were observed with the Ser304Ala mutant. The regio- and enantioselective oxidation of an analogue of metoprolol, in which the hydroxyl group attached to the chiral carbon was replaced by a methyl moiety, was again identical with both wild-type and Ser304Ala mutant. However, the observed selectivity was the reverse of that observed with metoprolol. Collectively, these data indicate that Ser304 is unlikely to be a key ligand-binding residue, although the residue may indeed be located in the active-site cavity. The reversal of selectivity with the methyl analogue of metoprolol indicates that the hydroxyl group attached to the chiral centre of ligands, such as metoprolol, is important in defining the enzyme's selective properties, and that a hydrogen-bonding residue, other than Ser304, may be involved in this interaction. Current homology models of the active site of CYP2D6 that predict a hydrogen-bond interaction between Ser304 and specific ligands will need to be re-evaluated, and other candidate residues capable of such an interaction nominated and tested by site-directed mutagenesis studies.
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February 2000
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Research Article|
January 25 2000
Evidence that serine 304 is not a key ligand-binding residue in the active site of cytochrome P450 2D6
S. Wynne ELLIS;
S. Wynne ELLIS
1
*Section of Molecular Pharmacology and Pharmacogenetics, L Floor, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, U.K.
1To whom correspondence should be addressed (e-mail S.Ellis@;Sheffield.ac.uk).
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Graham P. HAYHURST;
Graham P. HAYHURST
*Section of Molecular Pharmacology and Pharmacogenetics, L Floor, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, U.K.
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Tracy LIGHTFOOT;
Tracy LIGHTFOOT
*Section of Molecular Pharmacology and Pharmacogenetics, L Floor, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, U.K.
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Gillian SMITH;
Gillian SMITH
†Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, U.K.
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Jacky HARLOW;
Jacky HARLOW
*Section of Molecular Pharmacology and Pharmacogenetics, L Floor, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, U.K.
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Karen ROWLAND-YEO;
Karen ROWLAND-YEO
*Section of Molecular Pharmacology and Pharmacogenetics, L Floor, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, U.K.
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Cecilia LARSSON;
Cecilia LARSSON
*Section of Molecular Pharmacology and Pharmacogenetics, L Floor, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, U.K.
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Jurgen MAHLING;
Jurgen MAHLING
‡Department of Chemistry, Dainton Building, University of Sheffield, Sheffield S3 7HF, U.K.
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Chang K. LIM;
Chang K. LIM
§MRC Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Leicester LE1 9HN, U.K.
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C. Roland WOLF;
C. Roland WOLF
†Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee DD1 9SY, U.K.
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Michael G. BLACKBURN;
Michael G. BLACKBURN
‡Department of Chemistry, Dainton Building, University of Sheffield, Sheffield S3 7HF, U.K.
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Martin S. LENNARD;
Martin S. LENNARD
*Section of Molecular Pharmacology and Pharmacogenetics, L Floor, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, U.K.
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Geoffrey T. TUCKER
Geoffrey T. TUCKER
*Section of Molecular Pharmacology and Pharmacogenetics, L Floor, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, U.K.
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Publisher: Portland Press Ltd
Received:
July 29 1999
Revision Received:
October 08 1999
Accepted:
November 23 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 345 (3): 565–571.
Article history
Received:
July 29 1999
Revision Received:
October 08 1999
Accepted:
November 23 1999
Citation
S. Wynne ELLIS, Graham P. HAYHURST, Tracy LIGHTFOOT, Gillian SMITH, Jacky HARLOW, Karen ROWLAND-YEO, Cecilia LARSSON, Jurgen MAHLING, Chang K. LIM, C. Roland WOLF, Michael G. BLACKBURN, Martin S. LENNARD, Geoffrey T. TUCKER; Evidence that serine 304 is not a key ligand-binding residue in the active site of cytochrome P450 2D6. Biochem J 1 February 2000; 345 (3): 565–571. doi: https://doi.org/10.1042/bj3450565
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