TY - JOUR
T1 - Phe120 contributes to the regiospecificity of cytochrome P450 2D6
T2 - Mutation leads to the formation of a novel dextromethorphan metabolite
AU - Flanagan, Jack U.
AU - Maréchal, Jean Didier
AU - Ward, Richard
AU - Kemp, Carol A.
AU - McLaughlin, Lesley A.
AU - Sutcliffe, Michael J.
AU - Roberts, Gordon C.K.
AU - Paine, Mark J.I.
AU - Wolf, C. Roland
PY - 2004/6/1
Y1 - 2004/6/1
N2 - Although the residues that determine the preference of CYP2D6 (cytochrome P450 2D6) for compounds containing a basic nitrogen are well characterized, the contribution of other active site residues to substrate binding and orientation is less well understood. Our structural model of CYP2D6 identifies the aromatic residue Phe120 as a likely major feature of the active site. To examine the role of Phe120, mutants of CYP2D6 in which this residue has been substituted by alanine, leucine, tyrosine, serine, histidine, tryptophan or methionine residues have been prepared in bacterial membranes co-expressing human cytochrome NADPH cytochrome P450 oxidoreductase. The mutants have been characterized using the prototypical bufuralol 1′ hydroxylase and dextromethorphan O- and N-demethylase activities of CYP2D6. Larger effects on Km values are observed for dextromethorphan O-demethylation than for bufuralol 1′ hydroxylation, indicating that the Phe120 side chain is more important in dextromethorphan than in bufuralol binding. A role for this side chain in determining the regiospecificity of substrate oxidation was indicated by changes in the relative rates of O- and N-demethylation of dextromethorphan and, notably, by the formation of 7-hydroxy dextromethrophan, a novel dextromethorphan metabolite, in mutants in which it had been substituted. Computational studies of dextromethorphan binding to the active site of the Phe120 → Ala mutant were carried out to throw light on the way in which the removal of this side chain leads to different modes of ligand binding.
AB - Although the residues that determine the preference of CYP2D6 (cytochrome P450 2D6) for compounds containing a basic nitrogen are well characterized, the contribution of other active site residues to substrate binding and orientation is less well understood. Our structural model of CYP2D6 identifies the aromatic residue Phe120 as a likely major feature of the active site. To examine the role of Phe120, mutants of CYP2D6 in which this residue has been substituted by alanine, leucine, tyrosine, serine, histidine, tryptophan or methionine residues have been prepared in bacterial membranes co-expressing human cytochrome NADPH cytochrome P450 oxidoreductase. The mutants have been characterized using the prototypical bufuralol 1′ hydroxylase and dextromethorphan O- and N-demethylase activities of CYP2D6. Larger effects on Km values are observed for dextromethorphan O-demethylation than for bufuralol 1′ hydroxylation, indicating that the Phe120 side chain is more important in dextromethorphan than in bufuralol binding. A role for this side chain in determining the regiospecificity of substrate oxidation was indicated by changes in the relative rates of O- and N-demethylation of dextromethorphan and, notably, by the formation of 7-hydroxy dextromethrophan, a novel dextromethorphan metabolite, in mutants in which it had been substituted. Computational studies of dextromethorphan binding to the active site of the Phe120 → Ala mutant were carried out to throw light on the way in which the removal of this side chain leads to different modes of ligand binding.
KW - Cytochrome P450
KW - Cytochrome P450 2D6 (CYP2D6)
KW - Dextromethorphan
KW - Drug metabolism
KW - Ligand-binding property
KW - Mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=5044245324&partnerID=8YFLogxK
U2 - 10.1042/BJ20040062
DO - 10.1042/BJ20040062
M3 - Article
C2 - 14992686
AN - SCOPUS:5044245324
SN - 0264-6021
VL - 380
SP - 353
EP - 360
JO - Biochemical Journal
JF - Biochemical Journal
IS - 2
ER -