Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Jun 1;316(Pt 2):647–654. doi: 10.1042/bj3160647

Influence of amino acid residue 374 of cytochrome P-450 2D6 (CYP2D6) on the regio- and enantio-selective metabolism of metoprolol.

S W Ellis 1, K Rowland 1, M J Ackland 1, E Rekka 1, A P Simula 1, M S Lennard 1, C R Wolf 1, G T Tucker 1
PMCID: PMC1217396  PMID: 8687412

Abstract

Cytochrome P-450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme responsible for the oxidation of more than 30 widely used therapeutic agents. The enzymes encoded by the published genomic [Kimura, Umeno, Skoda, Meyer and Gonzalez (1989) Am. J. Hum. Genet. 45, 889-904] and cDNA [Gonzalez, Skoda, Kimura, Umeno, Zanger, Nebert, Gelboin, Hardwick and Meyer (1988) Nature 331, 442-446] sequences of CYP2D6, and presumed to represent wild-type sequences, differ at residue 374 and encode valine (CYP2D6-Val) and methionine (CYP2D6-Met) respectively. The influence of this amino acid difference on cytochrome P-450 expression, ligand binding, catalysis and stereoselective oxidation of metoprolol was investigated by the heterologous expression of the corresponding cDNAs in the yeast Saccharomyces cerevisiae. The level of expression of apo- and holo-protein was similar with each form of CYP2D6 cDNA, and the binding affinities of a series of ligands to CYP2D6-Val and CYP2D6-Met were identical. The enantioselective O-demethylation and alpha-hydroxylation of metoprolol were also similar with each form of CYP2D6, O-demethylation being R-(+)- enantioselective (CYP2D6-Val: R/S, 1.6; CYP2D6-Met: R/S, 1.4), whereas alpha-hydroxylation showed a preference for S-(-)-metoprolol (CYP2D6-Val: R/S, 0.7; CYP2D6-Met: R/S, 0.8). However, although the favoured regiomer overall was O-demethylmetoprolol (ODM), the regioselectivity for O-demethylation of each metoprolol enantiomer was significantly greater for CYP2D6-Val [R-(+)-: ODM/alpha-hydroxymetoprolol (alpha OH), 5.9; S-(-)-: ODM/alpha OH, 2.5) than that observed for CYP2D6-Met [R-(+)-: ODM/alpha OH, 2.2; S-(-)-: ODM/alpha OH, 1.4]. The stereoselective properties of CYP2D6-Val were consistent with those observed for CYP2D6 in human liver microsomes. The difference in the stereoselective properties of CYP2D6-Val and CYP2D6-Met were rationalized with respect to a homology model of the active site of CYP2D6 based on an alignment with the crystal structure of the bacterial cytochrome P-450BM-3' CYP102.

Full Text

The Full Text of this article is available as a PDF (602.1 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. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  3. Broly F., Gaedigk A., Heim M., Eichelbaum M., Morike K., Meyer U. A. Debrisoquine/sparteine hydroxylation genotype and phenotype: analysis of common mutations and alleles of CYP2D6 in a European population. DNA Cell Biol. 1991 Oct;10(8):545–558. doi: 10.1089/dna.1991.10.545. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Eichelbaum M., Gross A. S. The genetic polymorphism of debrisoquine/sparteine metabolism--clinical aspects. Pharmacol Ther. 1990;46(3):377–394. doi: 10.1016/0163-7258(90)90025-w. [DOI] [PubMed] [Google Scholar]
  6. Eichelbaum M., Spannbrucker N., Steincke B., Dengler H. J. Defective N-oxidation of sparteine in man: a new pharmacogenetic defect. Eur J Clin Pharmacol. 1979 Sep;16(3):183–187. doi: 10.1007/BF00562059. [DOI] [PubMed] [Google Scholar]
  7. Ellis S. W., Ching M. S., Watson P. F., Henderson C. J., Simula A. P., Lennard M. S., Tucker G. T., Woods H. F. Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast. Biochem Pharmacol. 1992 Aug 18;44(4):617–620. doi: 10.1016/0006-2952(92)90394-x. [DOI] [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. Evert B., Griese E. U., Eichelbaum M. A missense mutation in exon 6 of the CYP2D6 gene leading to a histidine 324 to proline exchange is associated with the poor metabolizer phenotype of sparteine. Naunyn Schmiedebergs Arch Pharmacol. 1994 Oct;350(4):434–439. doi: 10.1007/BF00178963. [DOI] [PubMed] [Google Scholar]
  10. Gonzalez F. J., Meyer U. A. Molecular genetics of the debrisoquin-sparteine polymorphism. Clin Pharmacol Ther. 1991 Sep;50(3):233–238. doi: 10.1038/clpt.1991.131. [DOI] [PubMed] [Google Scholar]
  11. Gonzalez F. J., Skoda R. C., Kimura S., Umeno M., Zanger U. M., Nebert D. W., Gelboin H. V., Hardwick J. P., Meyer U. A. Characterization of the common genetic defect in humans deficient in debrisoquine metabolism. Nature. 1988 Feb 4;331(6155):442–446. doi: 10.1038/331442a0. [DOI] [PubMed] [Google Scholar]
  12. Gotoh O. Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J Biol Chem. 1992 Jan 5;267(1):83–90. [PubMed] [Google Scholar]
  13. Gough A. C., Miles J. S., Spurr N. K., Moss J. E., Gaedigk A., Eichelbaum M., Wolf C. R. Identification of the primary gene defect at the cytochrome P450 CYP2D locus. Nature. 1990 Oct 25;347(6295):773–776. doi: 10.1038/347773a0. [DOI] [PubMed] [Google Scholar]
  14. Hasemann C. A., Kurumbail R. G., Boddupalli S. S., Peterson J. A., Deisenhofer J. Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure. 1995 Jan 15;3(1):41–62. doi: 10.1016/s0969-2126(01)00134-4. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Islam S. A., Wolf C. R., Lennard M. S., Sternberg M. J. A three-dimensional molecular template for substrates of human cytochrome P450 involved in debrisoquine 4-hydroxylation. Carcinogenesis. 1991 Dec;12(12):2211–2219. doi: 10.1093/carcin/12.12.2211. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kim M., Shen D. D., Eddy A. C., Nelson W. L., Roskos L. K. Inhibition of the enantioselective oxidative metabolism of metoprolol by verapamil in human liver microsomes. Drug Metab Dispos. 1993 Mar-Apr;21(2):309–317. [PubMed] [Google Scholar]
  19. Kimura S., Umeno M., Skoda R. C., Meyer U. A., Gonzalez F. J. The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene. Am J Hum Genet. 1989 Dec;45(6):889–904. [PMC free article] [PubMed] [Google Scholar]
  20. Korzekwa K. R., Jones J. P. Predicting the cytochrome P450 mediated metabolism of xenobiotics. Pharmacogenetics. 1993 Feb;3(1):1–18. doi: 10.1097/00008571-199302000-00001. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Koymans L., Vermeulen N. P., van Acker S. A., te Koppele J. M., Heykants J. J., Lavrijsen K., Meuldermans W., Donné-Op den Kelder G. M. A predictive model for substrates of cytochrome P450-debrisoquine (2D6). Chem Res Toxicol. 1992 Mar-Apr;5(2):211–219. doi: 10.1021/tx00026a010. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Mahgoub A., Idle J. R., Dring L. G., Lancaster R., Smith R. L. Polymorphic hydroxylation of Debrisoquine in man. Lancet. 1977 Sep 17;2(8038):584–586. doi: 10.1016/s0140-6736(77)91430-1. [DOI] [PubMed] [Google Scholar]
  25. Mautz D. S., Nelson W. L., Shen D. D. Regioselective and stereoselective oxidation of metoprolol and bufuralol catalyzed by microsomes containing cDNA-expressed human P4502D6. Drug Metab Dispos. 1995 Apr;23(4):513–517. [PubMed] [Google Scholar]
  26. Meyer U. A., Gut J., Kronbach T., Skoda C., Meier U. T., Catin T., Dayer P. The molecular mechanisms of two common polymorphisms of drug oxidation--evidence for functional changes in cytochrome P-450 isozymes catalysing bufuralol and mephenytoin oxidation. Xenobiotica. 1986 May;16(5):449–464. doi: 10.3109/00498258609050251. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Ouzounis C. A., Melvin W. T. Primary and secondary structural patterns in eukaryotic cytochrome P-450 families correspond to structures of the helix-rich domain of Pseudomonas putida cytochrome P-450cam. Indications for a similar overall topology. Eur J Biochem. 1991 Jun 1;198(2):307–315. doi: 10.1111/j.1432-1033.1991.tb16017.x. [DOI] [PubMed] [Google Scholar]
  30. Poulos T. L., Finzel B. C., Howard A. J. High-resolution crystal structure of cytochrome P450cam. J Mol Biol. 1987 Jun 5;195(3):687–700. doi: 10.1016/0022-2836(87)90190-2. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Saxena R., Shaw G. L., Relling M. V., Frame J. N., Moir D. T., Evans W. E., Caporaso N., Weiffenbach B. Identification of a new variant CYP2D6 allele with a single base deletion in exon 3 and its association with the poor metabolizer phenotype. Hum Mol Genet. 1994 Jun;3(6):923–926. doi: 10.1093/hmg/3.6.923. [DOI] [PubMed] [Google Scholar]
  33. Shaw L., Lennard M. S., Tucker G. T., Bax N. D., Woods H. F. Irreversible binding and metabolism of propranolol by human liver microsomes--relationship to polymorphic oxidation. Biochem Pharmacol. 1987 Jul 15;36(14):2283–2288. doi: 10.1016/0006-2952(87)90592-2. [DOI] [PubMed] [Google Scholar]
  34. Tucker G. T. Clinical implications of genetic polymorphism in drug metabolism. J Pharm Pharmacol. 1994 May;46 (Suppl 1):417–424. [PubMed] [Google Scholar]
  35. Tucker G. T., Lennard M. S., Ellis S. W., Woods H. F., Cho A. K., Lin L. Y., Hiratsuka A., Schmitz D. A., Chu T. Y. The demethylenation of methylenedioxymethamphetamine ("ecstasy") by debrisoquine hydroxylase (CYP2D6). Biochem Pharmacol. 1994 Mar 29;47(7):1151–1156. doi: 10.1016/0006-2952(94)90386-7. [DOI] [PubMed] [Google Scholar]
  36. Tucker G. T., Silas J. H., Iyun A. O., Lennard M. S., Smith A. J. Polymorphic hydroxylation of debrisoquine. Lancet. 1977 Oct 1;2(8040):718–718. doi: 10.1016/s0140-6736(77)90527-x. [DOI] [PubMed] [Google Scholar]
  37. Williams M. L., Lennard M. S., Martin I. J., Tucker G. T. Interindividual variation in the isomerization of 4-hydroxytamoxifen by human liver microsomes: involvement of cytochromes P450. Carcinogenesis. 1994 Dec;15(12):2733–2738. doi: 10.1093/carcin/15.12.2733. [DOI] [PubMed] [Google Scholar]
  38. Wolff T., Distlerath L. M., Worthington M. T., Groopman J. D., Hammons G. J., Kadlubar F. F., Prough R. A., Martin M. V., Guengerich F. P. Substrate specificity of human liver cytochrome P-450 debrisoquine 4-hydroxylase probed using immunochemical inhibition and chemical modeling. Cancer Res. 1985 May;45(5):2116–2122. [PubMed] [Google Scholar]

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

RESOURCES