Binding of bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine to wild-type and F120A mutant cytochrome P450 2D6 studied by resonance Raman spectroscopy

2006 ◽  
Vol 343 (3) ◽  
pp. 772-779 ◽  
Author(s):  
Alois Bonifacio ◽  
Peter H.J. Keizers ◽  
Jan N.M. Commandeur ◽  
Nico P.E. Vermeulen ◽  
Bruno Robert ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Cytochrome P450 ◽  
Resonance Raman Spectroscopy ◽  
Resonance Raman ◽  
Cytochrome P450 2D6 ◽  
Wild Type ◽  
P450 2D6

Surface-Enhanced Resonance Raman Scattering of Cytochrome P450-2D6 on Coated Silver Hydrosols

Langmuir ◽  
2007 ◽  
Vol 23 (4) ◽  
pp. 1860-1866 ◽  
Author(s):  
Alois Bonifacio ◽  
Peter H. J. Keizers ◽  
Nico P. E. Vermeulen ◽  
Jan N. M. Commandeur ◽  
Cees Gooijer ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Raman Scattering ◽  
Resonance Raman ◽  
Cytochrome P450 2D6 ◽  
Resonance Raman Scattering ◽  
Surface Enhanced ◽  
P450 2D6

Influence of phenylalanine-481 substitutions on the catalytic activity of cytochrome P450 2D6

2001 ◽  
Vol 355 (2) ◽  
pp. 373-379 ◽  
Author(s):  
Graham P. HAYHURST ◽  
Jacky HARLOW ◽  
Joey CHOWDRY ◽  
Esme GROSS ◽  
Emma HILTON ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Ligand Binding ◽  
Active Site ◽  
Dominant Role ◽  
Yeast Cells ◽  
Primary Role ◽  
Cytochrome P450 2D6 ◽  
Wild Type ◽  
Binding Residue ◽  
P450 2D6

Homology models of the active site of cytochrome P450 2D6 (CYP2D6) have identified phenylalanine 481 (Phe481) as a putative ligand-binding residue, its aromatic side chain being potentially capable of participating in π-π interactions with the benzene ring of ligands. We have tested this hypothesis by replacing Phe481 with tyrosine (Phe481 → Tyr), a conservative substitution, and with leucine (Phe481 → Leu) or glycine (Phe481 → Gly), two non-aromatic residues, and have compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived protein. The Phe481 → Tyr substitution did not alter the kinetics [Km (µM) and Vmax (pmol/min per pmol) respectively] of oxidation of S-metoprolol (27; 4.60), debrisoquine (46; 2.46) or dextromethorphan (2; 8.43) relative to the respective wild-type values [S-metoprolol (26; 3.48), debrisoquine (51; 3.20) and dextromethorphan (2; 8.16)]. The binding capacities [Ks (µM)] of a range of CYP2D6 ligands to the Phe481 → Tyr enzyme (S-metoprolol, 22.8; debrisoquine, 12.5; dextromethorphan, 2.3; quinidine, 0.13) were also similar to those for the wild-type enzyme (S-metoprolol, 10.9; debrisoquine, 8.9; dextromethorphan, 3.1; quinidine, 0.10). In contrast, the Phe481 → Leu and Phe481 → Gly substitutions increased significantly (3-16-fold) the Km values of oxidation of the three substrates [S-metoprolol (120-124µM), debrisoquine (152-184µM) and dextromethorphan (20-31µM)]. Similarly, the Ks values of the ligands to Phe481 → Leu and Phe481 → Gly mutants were also increased 3 to 10-fold (S-metoprolol, 33.2-41.9µM; debrisoquine, 85-90µM; dextromethorphan, 15.7-18.8µM; quinidine 0.35-0.53µM). However, contrary to a recent proposal that Phe481 has the dominant role in the binding of substrates that undergo CYP2D6-mediated N-dealkylation routes of metabolism, the Phe481 → Gly substitution did not substantially decrease the capacity of the enzyme to N-deisopropylate metoprolol (wild-type, 1.12pmol/min per pmol of P450; Phe481 → Gly, 0.71), whereas an Asp301 → Gly substitution decreased the N-dealkylation reaction by 95% of the wild-type rate. Overall, our results are consistent with the proposal that Phe481 is a ligand-binding residue in the active site of CYP2D6 and that the residue interacts with ligands via a π-π interaction between its phenyl ring and the aromatic moiety of the ligand. However, the relative importance of Phe481 in binding is ligand-dependent; furthermore, its importance is secondary to that of Asp301. Finally, contrary to predictions of a recent homology model, Phe481 does not seem to have a primary role in CYP2D6-mediated N-dealkylation.

scholarly journals Free energies of binding of R- and S-propranolol to wild-type and F483A mutant cytochrome P450 2D6 from molecular dynamics simulations

2007 ◽  
Vol 36 (6) ◽  
pp. 589-599 ◽  
Author(s):  
Chris de Graaf ◽  
Chris Oostenbrink ◽  
Peter H. J. Keizers ◽  
Barbara M. A. van Vugt-Lussenburg ◽  
Jan N. M. Commandeur ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Cytochrome P450 ◽  
Molecular Dynamics Simulations ◽  
Cytochrome P450 2D6 ◽  
Free Energies ◽  
Wild Type ◽  
Dynamics Simulations ◽  
P450 2D6
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