Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Apr 15;355(Pt 2):373–379. doi: 10.1042/0264-6021:3550373

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

G P Hayhurst 1, J Harlow 1, J Chowdry 1, E Gross 1, E Hilton 1, M S Lennard 1, G T Tucker 1, S W Ellis 1
PMCID: PMC1221748  PMID: 11284724

Abstract

Homology models of the active site of cytochrome P450 2D6 (CYP2D6) have identified phenylalanine 481 (Phe(481)) as a putative ligand-binding residue, its aromatic side chain being potentially capable of participating in pi-pi interactions with the benzene ring of ligands. We have tested this hypothesis by replacing Phe(481) with tyrosine (Phe(481)-->Tyr), a conservative substitution, and with leucine (Phe(481)-->Leu) or glycine (Phe(481)-->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 Phe(481)-->Tyr substitution did not alter the kinetics [K(m) (microM) and V(max) (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 [K(s) (microM)] of a range of CYP2D6 ligands to the Phe(481)-->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 Phe(481)-->Leu and Phe(481)-->Gly substitutions increased significantly (3-16-fold) the K(m) values of oxidation of the three substrates [S-metoprolol (120-124 microM), debrisoquine (152-184 microM) and dextromethorphan (20-31 microM)]. Similarly, the K(s) values of the ligands to Phe(481)-->Leu and Phe(481)-->Gly mutants were also increased 3 to 10-fold (S-metoprolol, 33.2-41.9 microM; debrisoquine, 85-90 microM; dextromethorphan, 15.7-18.8 microM; quinidine 0.35-0.53 microM). However, contrary to a recent proposal that Phe(481) has the dominant role in the binding of substrates that undergo CYP2D6-mediated N-dealkylation routes of metabolism, the Phe(481)-->Gly substitution did not substantially decrease the capacity of the enzyme to N-deisopropylate metoprolol (wild-type, 1.12 pmol/min per pmol of P450; Phe(481)-->Gly, 0.71), whereas an Asp(301)-->Gly substitution decreased the N-dealkylation reaction by 95% of the wild-type rate. Overall, our results are consistent with the proposal that Phe(481) is a ligand-binding residue in the active site of CYP2D6 and that the residue interacts with ligands via a pi-pi interaction between its phenyl ring and the aromatic moiety of the ligand. However, the relative importance of Phe(481) in binding is ligand-dependent; furthermore, its importance is secondary to that of Asp(301). Finally, contrary to predictions of a recent homology model, Phe(481) does not seem to have a primary role in CYP2D6-mediated N-dealkylation.

Full Text

The Full Text of this article is available as a PDF (143.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Becker D. M., Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182–187. doi: 10.1016/0076-6879(91)94015-5. [DOI] [PubMed] [Google Scholar]
  2. Chen Z. R., Somogyi A. A., Bochner F. Simultaneous determination of dextromethorphan and three metabolites in plasma and urine using high-performance liquid chromatography with application to their disposition in man. Ther Drug Monit. 1990 Jan;12(1):97–104. doi: 10.1097/00007691-199001000-00018. [DOI] [PubMed] [Google Scholar]
  3. Cupp-Vickery J. R., Poulos T. L. Structure of cytochrome P450eryF involved in erythromycin biosynthesis. Nat Struct Biol. 1995 Feb;2(2):144–153. doi: 10.1038/nsb0295-144. [DOI] [PubMed] [Google Scholar]
  4. Daly A. K., Brockmöller J., Broly F., Eichelbaum M., Evans W. E., Gonzalez F. J., Huang J. D., Idle J. R., Ingelman-Sundberg M., Ishizaki T. Nomenclature for human CYP2D6 alleles. Pharmacogenetics. 1996 Jun;6(3):193–201. doi: 10.1097/00008571-199606000-00001. [DOI] [PubMed] [Google Scholar]
  5. De Groot M. J., Vermeulen N. P. Modeling the active sites of cytochrome P450s and glutathione S-transferases, two of the most important biotransformation enzymes. Drug Metab Rev. 1997 Aug;29(3):747–799. doi: 10.3109/03602539709037596. [DOI] [PubMed] [Google Scholar]
  6. Ekins S., Bravi G., Binkley S., Gillespie J. S., Ring B. J., Wikel J. H., Wrighton S. A. Three and four dimensional-quantitative structure activity relationship (3D/4D-QSAR) analyses of CYP2D6 inhibitors. Pharmacogenetics. 1999 Aug;9(4):477–489. [PubMed] [Google Scholar]
  7. Ellis S. W., Hayhurst G. P., Lightfoot T., Smith G., Harlow J., Rowland-Yeo K., Larsson C., Mahling J., Lim C. K., Wolf C. R. Evidence that serine 304 is not a key ligand-binding residue in the active site of cytochrome P450 2D6. Biochem J. 2000 Feb 1;345(Pt 3):565–571. [PMC free article] [PubMed] [Google Scholar]
  8. Ellis S. W., Hayhurst G. P., Smith G., Lightfoot T., Wong M. M., Simula A. P., Ackland M. J., Sternberg M. J., Lennard M. S., Tucker G. T. Evidence that aspartic acid 301 is a critical substrate-contact residue in the active site of cytochrome P450 2D6. J Biol Chem. 1995 Dec 8;270(49):29055–29058. doi: 10.1074/jbc.270.49.29055. [DOI] [PubMed] [Google Scholar]
  9. Ellis S. W., Rowland K., Ackland M. J., Rekka E., Simula A. P., Lennard M. S., Wolf C. R., Tucker G. T. Influence of amino acid residue 374 of cytochrome P-450 2D6 (CYP2D6) on the regio- and enantio-selective metabolism of metoprolol. Biochem J. 1996 Jun 1;316(Pt 2):647–654. doi: 10.1042/bj3160647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hasemann C. A., Ravichandran K. G., Peterson J. A., Deisenhofer J. Crystal structure and refinement of cytochrome P450terp at 2.3 A resolution. J Mol Biol. 1994 Mar 4;236(4):1169–1185. doi: 10.1016/0022-2836(94)90019-1. [DOI] [PubMed] [Google Scholar]
  11. Jefcoate C. R. Measurement of substrate and inhibitor binding to microsomal cytochrome P-450 by optical-difference spectroscopy. Methods Enzymol. 1978;52:258–279. doi: 10.1016/s0076-6879(78)52029-6. [DOI] [PubMed] [Google Scholar]
  12. Koymans L. M., Vermeulen N. P., Baarslag A., Donné-Op den Kelder G. M. A preliminary 3D model for cytochrome P450 2D6 constructed by homology model building. J Comput Aided Mol Des. 1993 Jun;7(3):281–289. doi: 10.1007/BF00125503. [DOI] [PubMed] [Google Scholar]
  13. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Lewis D. F., Dickins M., Eddershaw P. J., Tarbit M. H., Goldfarb P. S. Cytochrome P450 substrate specificities, substrate structural templates and enzyme active site geometries. Drug Metabol Drug Interact. 1999;15(1):1–49. doi: 10.1515/dmdi.1999.15.1.1. [DOI] [PubMed] [Google Scholar]
  16. Lewis D. F., Eddershaw P. J., Goldfarb P. S., Tarbit M. H. Molecular modelling of cytochrome P4502D6 (CYP2D6) based on an alignment with CYP102: structural studies on specific CYP2D6 substrate metabolism. Xenobiotica. 1997 Apr;27(4):319–339. doi: 10.1080/004982597240497. [DOI] [PubMed] [Google Scholar]
  17. Lewis D. F. Three-dimensional models of human and other mammalian microsomal P450s constructed from an alignment with P450102 (P450bm3). Xenobiotica. 1995 Apr;25(4):333–366. doi: 10.3109/00498259509061857. [DOI] [PubMed] [Google Scholar]
  18. Lightfoot T., Ellis S. W., Mahling J., Ackland M. J., Blaney F. E., Bijloo G. J., De Groot M. J., Vermeulen N. P., Blackburn G. M., Lennard M. S. Regioselective hydroxylation of debrisoquine by cytochrome P4502D6: implications for active site modelling. Xenobiotica. 2000 Mar;30(3):219–233. doi: 10.1080/004982500237622. [DOI] [PubMed] [Google Scholar]
  19. Marez D., Legrand M., Sabbagh N., Lo Guidice J. M., Spire C., Lafitte J. J., Meyer U. A., Broly F. Polymorphism of the cytochrome P450 CYP2D6 gene in a European population: characterization of 48 mutations and 53 alleles, their frequencies and evolution. Pharmacogenetics. 1997 Jun;7(3):193–202. doi: 10.1097/00008571-199706000-00004. [DOI] [PubMed] [Google Scholar]
  20. Modi S., Paine M. J., Sutcliffe M. J., Lian L. Y., Primrose W. U., Wolf C. R., Roberts G. C. A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding. Biochemistry. 1996 Apr 9;35(14):4540–4550. doi: 10.1021/bi952742o. [DOI] [PubMed] [Google Scholar]
  21. Narimatsu S., Kato R., Horie T., Ono S., Tsutsui M., Yabusaki Y., Ohmori S., Kitada M., Ichioka T., Shimada N. Enantioselectivity of bunitrolol 4-hydroxylation is reversed by the change of an amino acid residue from valine to methionine at position 374 of cytochrome P450-2D6. Chirality. 1999;11(1):1–9. doi: 10.1002/(SICI)1520-636X(1999)11:1<1::AID-CHIR1>3.0.CO;2-E. [DOI] [PubMed] [Google Scholar]
  22. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. J Biol Chem. 1964 Jul;239:2370–2378. [PubMed] [Google Scholar]
  23. Otton S. V., Crewe H. K., Lennard M. S., Tucker G. T., Woods H. F. Use of quinidine inhibition to define the role of the sparteine/debrisoquine cytochrome P450 in metoprolol oxidation by human liver microsomes. J Pharmacol Exp Ther. 1988 Oct;247(1):242–247. [PubMed] [Google Scholar]
  24. Poulos T. L., Finzel B. C., Gunsalus I. C., Wagner G. C., Kraut J. The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. J Biol Chem. 1985 Dec 25;260(30):16122–16130. [PubMed] [Google Scholar]
  25. Ravichandran K. G., Boddupalli S. S., Hasermann C. A., Peterson J. A., Deisenhofer J. Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's. Science. 1993 Aug 6;261(5122):731–736. doi: 10.1126/science.8342039. [DOI] [PubMed] [Google Scholar]
  26. Smith G., Modi S., Pillai I., Lian L. Y., Sutcliffe M. J., Pritchard M. P., Friedberg T., Roberts G. C., Wolf C. R. Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity. Biochem J. 1998 May 1;331(Pt 3):783–792. doi: 10.1042/bj3310783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Strobl G. R., von Kruedener S., Stöckigt J., Guengerich F. P., Wolff T. Development of a pharmacophore for inhibition of human liver cytochrome P-450 2D6: molecular modeling and inhibition studies. J Med Chem. 1993 Apr 30;36(9):1136–1145. doi: 10.1021/jm00061a004. [DOI] [PubMed] [Google Scholar]
  28. Szklarz G. D., Halpert J. R. Use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships of mammalian cytochromes P450. Life Sci. 1997;61(26):2507–2520. doi: 10.1016/s0024-3205(97)00717-0. [DOI] [PubMed] [Google Scholar]
  29. Tucker G. T. Clinical implications of genetic polymorphism in drug metabolism. J Pharm Pharmacol. 1994 May;46 (Suppl 1):417–424. [PubMed] [Google Scholar]
  30. Williams P. A., Cosme J., Sridhar V., Johnson E. F., McRee D. E. Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol Cell. 2000 Jan;5(1):121–131. doi: 10.1016/s1097-2765(00)80408-6. [DOI] [PubMed] [Google Scholar]
  31. Wiseman H., Lewis D. F. The metabolism of tamoxifen by human cytochromes P450 is rationalized by molecular modelling of the enzyme-substrate interactions: potential importance to its proposed anti-carcinogenic/carcinogenic actions. Carcinogenesis. 1996 Jun;17(6):1357–1360. doi: 10.1093/carcin/17.6.1357. [DOI] [PubMed] [Google Scholar]
  32. de Groot M. J., Ackland M. J., Horne V. A., Alex A. A., Jones B. C. A novel approach to predicting P450 mediated drug metabolism. CYP2D6 catalyzed N-dealkylation reactions and qualitative metabolite predictions using a combined protein and pharmacophore model for CYP2D6. J Med Chem. 1999 Oct 7;42(20):4062–4070. doi: 10.1021/jm991058v. [DOI] [PubMed] [Google Scholar]
  33. de Groot M. J., Ackland M. J., Horne V. A., Alex A. A., Jones B. C. Novel approach to predicting P450-mediated drug metabolism: development of a combined protein and pharmacophore model for CYP2D6. J Med Chem. 1999 May 6;42(9):1515–1524. doi: 10.1021/jm981118h. [DOI] [PubMed] [Google Scholar]
  34. de Groot M. J., Vermeulen N. P., Kramer J. D., van Acker F. A., Donné-Op den Kelder G. M. A three-dimensional protein model for human cytochrome P450 2D6 based on the crystal structures of P450 101, P450 102, and P450 108. Chem Res Toxicol. 1996 Oct-Nov;9(7):1079–1091. doi: 10.1021/tx960003i. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES