Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Sep 15;374(Pt 3):731–737. doi: 10.1042/BJ20030625

Clarification of the role of key active site residues of glutathione transferase zeta/maleylacetoacetate isomerase by a new spectrophotometric technique.

Philip G Board 1, Matthew C Taylor 1, Marjorie Coggan 1, Michael W Parker 1, Hoffman B Lantum 1, M W Anders 1
PMCID: PMC1223650  PMID: 12852784

Abstract

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(+/-)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16-->Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the K(m) for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of alpha-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the alpha-halo acid substrates.

Full Text

The Full Text of this article is available as a PDF (123.0 KB).

Selected References

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

  1. Anandarajah K., Kiefer P. M., Jr, Donohoe B. S., Copley S. D. Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol. Biochemistry. 2000 May 9;39(18):5303–5311. doi: 10.1021/bi9923813. [DOI] [PubMed] [Google Scholar]
  2. Anderson W. B., Board P. G., Gargano B., Anders M. W. Inactivation of glutathione transferase zeta by dichloroacetic acid and other fluorine-lacking alpha-haloalkanoic acids. Chem Res Toxicol. 1999 Dec;12(12):1144–1149. doi: 10.1021/tx990085l. [DOI] [PubMed] [Google Scholar]
  3. Anderson Wayne B., Liebler Daniel C., Board Philip G., Anders M. W. Mass spectral characterization of dichloroacetic acid-modified human glutathione transferase zeta. Chem Res Toxicol. 2002 Nov;15(11):1387–1397. doi: 10.1021/tx025553x. [DOI] [PubMed] [Google Scholar]
  4. Armstrong R. N. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem Res Toxicol. 1997 Jan;10(1):2–18. doi: 10.1021/tx960072x. [DOI] [PubMed] [Google Scholar]
  5. Beutler E., Mathai C. K., Smith J. E. Biochemical variants of glucose-6-phosphate dehydrogenase giving rise to congenital nonspherocytic hemolytic disease. Blood. 1968 Feb;31(2):131–150. [PubMed] [Google Scholar]
  6. Björnestedt R., Stenberg G., Widersten M., Board P. G., Sinning I., Jones T. A., Mannervik B. Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1. J Mol Biol. 1995 Apr 7;247(4):765–773. doi: 10.1016/s0022-2836(05)80154-8. [DOI] [PubMed] [Google Scholar]
  7. Blackburn A. C., Tzeng H. F., Anders M. W., Board P. G. Discovery of a functional polymorphism in human glutathione transferase zeta by expressed sequence tag database analysis. Pharmacogenetics. 2000 Feb;10(1):49–57. doi: 10.1097/00008571-200002000-00007. [DOI] [PubMed] [Google Scholar]
  8. Board P. G., Baker R. T., Chelvanayagam G., Jermiin L. S. Zeta, a novel class of glutathione transferases in a range of species from plants to humans. Biochem J. 1997 Dec 15;328(Pt 3):929–935. doi: 10.1042/bj3280929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Board P. G., Coggan M., Chelvanayagam G., Easteal S., Jermiin L. S., Schulte G. K., Danley D. E., Hoth L. R., Griffor M. C., Kamath A. V. Identification, characterization, and crystal structure of the Omega class glutathione transferases. J Biol Chem. 2000 Aug 11;275(32):24798–24806. doi: 10.1074/jbc.M001706200. [DOI] [PubMed] [Google Scholar]
  10. Bull R. J., Sanchez I. M., Nelson M. A., Larson J. L., Lansing A. J. Liver tumor induction in B6C3F1 mice by dichloroacetate and trichloroacetate. Toxicology. 1990 Sep;63(3):341–359. doi: 10.1016/0300-483x(90)90195-m. [DOI] [PubMed] [Google Scholar]
  11. Caccuri A. M., Antonini G., Nicotra M., Battistoni A., Lo Bello M., Board P. G., Parker M. W., Ricci G. Catalytic mechanism and role of hydroxyl residues in the active site of theta class glutathione S-transferases. Investigation of Ser-9 and Tyr-113 in a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina. J Biol Chem. 1997 Nov 21;272(47):29681–29686. doi: 10.1074/jbc.272.47.29681. [DOI] [PubMed] [Google Scholar]
  12. DeAngelo A. B., Daniel F. B., Most B. M., Olson G. R. The carcinogenicity of dichloroacetic acid in the male Fischer 344 rat. Toxicology. 1996 Dec 18;114(3):207–221. doi: 10.1016/s0300-483x(96)03510-x. [DOI] [PubMed] [Google Scholar]
  13. Fernández-Cañn J. M., Peñalva M. A. Characterization of a fungal maleylacetoacetate isomerase gene and identification of its human homologue. J Biol Chem. 1998 Jan 2;273(1):329–337. doi: 10.1074/jbc.273.1.329. [DOI] [PubMed] [Google Scholar]
  14. Fernández-Cañn José Manuel, Baetscher Manfred W., Finegold Milton, Burlingame Terry, Gibson K. Michael, Grompe Markus. Maleylacetoacetate isomerase (MAAI/GSTZ)-deficient mice reveal a glutathione-dependent nonenzymatic bypass in tyrosine catabolism. Mol Cell Biol. 2002 Jul;22(13):4943–4951. doi: 10.1128/MCB.22.13.4943-4951.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harrington-Brock K., Doerr C. L., Moore M. M. Mutagenicity of three disinfection by-products: di- and trichloroacetic acid and chloral hydrate in L5178Y/TK +/- (-)3.7.2C mouse lymphoma cells. Mutat Res. 1998 Mar 30;413(3):265–276. doi: 10.1016/s1383-5718(98)00026-6. [DOI] [PubMed] [Google Scholar]
  16. Jemth P., Mannervik B. Active site serine promotes stabilization of the reactive glutathione thiolate in rat glutathione transferase T2-2. Evidence against proposed sulfatase activity of the corresponding human enzyme. J Biol Chem. 2000 Mar 24;275(12):8618–8624. doi: 10.1074/jbc.275.12.8618. [DOI] [PubMed] [Google Scholar]
  17. Kiefer Philip M., Jr, Copley Shelley D. Characterization of the initial steps in the reductive dehalogenation catalyzed by tetrachlorohydroquinone dehalogenase. Biochemistry. 2002 Jan 29;41(4):1315–1322. doi: 10.1021/bi0117504. [DOI] [PubMed] [Google Scholar]
  18. Kortemme T., Creighton T. E. Ionisation of cysteine residues at the termini of model alpha-helical peptides. Relevance to unusual thiol pKa values in proteins of the thioredoxin family. J Mol Biol. 1995 Nov 10;253(5):799–812. doi: 10.1006/jmbi.1995.0592. [DOI] [PubMed] [Google Scholar]
  19. Lantum Hoffman B. M., Board Philip G., Anders M. W. Kinetics of the biotransformation of maleylacetone and chlorofluoroacetic acid by polymorphic variants of human glutathione transferase zeta (hGSTZ1-1). Chem Res Toxicol. 2002 Jul;15(7):957–963. doi: 10.1021/tx010095y. [DOI] [PubMed] [Google Scholar]
  20. Oakley A. J., Lo Bello M., Battistoni A., Ricci G., Rossjohn J., Villar H. O., Parker M. W. The structures of human glutathione transferase P1-1 in complex with glutathione and various inhibitors at high resolution. J Mol Biol. 1997 Nov 21;274(1):84–100. doi: 10.1006/jmbi.1997.1364. [DOI] [PubMed] [Google Scholar]
  21. Polekhina G., Board P. G., Blackburn A. C., Parker M. W. Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basis for its remarkable catalytic promiscuity. Biochemistry. 2001 Feb 13;40(6):1567–1576. doi: 10.1021/bi002249z. [DOI] [PubMed] [Google Scholar]
  22. Polekhina G., Board P. G., Blackburn A. C., Parker M. W. Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basis for its remarkable catalytic promiscuity. Biochemistry. 2001 Feb 13;40(6):1567–1576. doi: 10.1021/bi002249z. [DOI] [PubMed] [Google Scholar]
  23. Rossjohn J., McKinstry W. J., Oakley A. J., Verger D., Flanagan J., Chelvanayagam G., Tan K. L., Board P. G., Parker M. W. Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site. Structure. 1998 Mar 15;6(3):309–322. doi: 10.1016/s0969-2126(98)00034-3. [DOI] [PubMed] [Google Scholar]
  24. Rossjohn J., Polekhina G., Feil S. C., Allocati N., Masulli M., Di Illio C., Parker M. W. A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications. Structure. 1998 Jun 15;6(6):721–734. doi: 10.1016/s0969-2126(98)00074-4. [DOI] [PubMed] [Google Scholar]
  25. Seltzer S. Purification and properties of maleylacetone cis-trans isomerase from vibrio 01. J Biol Chem. 1973 Jan 10;248(1):215–222. [PubMed] [Google Scholar]
  26. Sinning I., Kleywegt G. J., Cowan S. W., Reinemer P., Dirr H. W., Huber R., Gilliland G. L., Armstrong R. N., Ji X., Board P. G. Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes. J Mol Biol. 1993 Jul 5;232(1):192–212. doi: 10.1006/jmbi.1993.1376. [DOI] [PubMed] [Google Scholar]
  27. Stacpoole P. W., Henderson G. N., Yan Z., James M. O. Clinical pharmacology and toxicology of dichloroacetate. Environ Health Perspect. 1998 Aug;106 (Suppl 4):989–994. doi: 10.1289/ehp.98106s4989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tan K. L., Chelvanayagam G., Parker M. W., Board P. G. Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues. Biochem J. 1996 Oct 1;319(Pt 1):315–321. doi: 10.1042/bj3190315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tanguay R. M., Jorquera R., Poudrier J., St-Louis M. Tyrosine and its catabolites: from disease to cancer. Acta Biochim Pol. 1996;43(1):209–216. [PubMed] [Google Scholar]
  30. Tao L., Kramer P. M., Ge R., Pereira M. A. Effect of dichloroacetic acid and trichloroacetic acid on DNA methylation in liver and tumors of female B6C3F1 mice. Toxicol Sci. 1998 Jun;43(2):139–144. doi: 10.1006/toxs.1998.2449. [DOI] [PubMed] [Google Scholar]
  31. Thom R., Dixon D. P., Edwards R., Cole D. J., Lapthorn A. J. The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism. J Mol Biol. 2001 May 18;308(5):949–962. doi: 10.1006/jmbi.2001.4638. [DOI] [PubMed] [Google Scholar]
  32. Tong Z., Board P. G., Anders M. W. Glutathione transferase zeta catalyses the oxygenation of the carcinogen dichloroacetic acid to glyoxylic acid. Biochem J. 1998 Apr 15;331(Pt 2):371–374. doi: 10.1042/bj3310371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tong Z., Board P. G., Anders M. W. Glutathione transferase zeta-catalyzed biotransformation of dichloroacetic acid and other alpha-haloacids. Chem Res Toxicol. 1998 Nov;11(11):1332–1338. doi: 10.1021/tx980144f. [DOI] [PubMed] [Google Scholar]
  34. Tzeng H. F., Blackburn A. C., Board P. G., Anders M. W. Polymorphism- and species-dependent inactivation of glutathione transferase zeta by dichloroacetate. Chem Res Toxicol. 2000 Apr;13(4):231–236. doi: 10.1021/tx990175q. [DOI] [PubMed] [Google Scholar]
  35. Wilce M. C., Board P. G., Feil S. C., Parker M. W. Crystal structure of a theta-class glutathione transferase. EMBO J. 1995 May 15;14(10):2133–2143. doi: 10.1002/j.1460-2075.1995.tb07207.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES