Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Nov 1;343(Pt 3):525–531.

The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases.

C S Allardyce 1, P D McDonagh 1, L Y Lian 1, C R Wolf 1, G C Roberts 1
PMCID: PMC1220582  PMID: 10527929

Abstract

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the 'essential' Tyr(9) by phenylalanine leads to a marked decrease in the k(cat) for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on k(cat) for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in k(cat) for CDNB, but an increase in k(cat) for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity ('turnover number') for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

Full Text

The Full Text of this article is available as a PDF (169.0 KB).

Selected References

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

  1. Adang A. E., Brussee J., Meyer D. J., Coles B., Ketterer B., van der Gen A., Mulder G. J. Substrate specificity of rat liver glutathione S-transferase isoenzymes for a series of glutathione analogues, modified at the gamma-glutamyl moiety. Biochem J. 1988 Oct 15;255(2):721–724. [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong R. N. Glutathione S-transferases: structure and mechanism of an archetypical detoxication enzyme. Adv Enzymol Relat Areas Mol Biol. 1994;69:1–44. doi: 10.1002/9780470123157.ch1. [DOI] [PubMed] [Google Scholar]
  3. Atkins W. M., Wang R. W., Bird A. W., Newton D. J., Lu A. Y. The catalytic mechanism of glutathione S-transferase (GST). Spectroscopic determination of the pKa of Tyr-9 in rat alpha 1-1 GST. J Biol Chem. 1993 Sep 15;268(26):19188–19191. [PubMed] [Google Scholar]
  4. Barycki J. J., Colman R. F. Affinity labeling of glutathione S-transferase, isozyme 4-4, by 4-(fluorosulfonyl)benzoic acid reveals Tyr115 to be an important determinant of xenobiotic substrate specificity. Biochemistry. 1993 Dec 7;32(48):13002–13011. doi: 10.1021/bi00211a008. [DOI] [PubMed] [Google Scholar]
  5. Barycki J. J., Colman R. F. Identification of the nonsubstrate steroid binding site of rat liver glutathione S-transferase, isozyme 1-1, by the steroid affinity label, 3beta-(iodoacetoxy)dehydroisoandrosterone. Arch Biochem Biophys. 1997 Sep 1;345(1):16–31. doi: 10.1006/abbi.1997.0244. [DOI] [PubMed] [Google Scholar]
  6. Board P. G., Mannervik B. The contribution of the C-terminal sequence to the catalytic activity of GST2, a human alpha-class glutathione transferase. Biochem J. 1991 Apr 1;275(Pt 1):171–174. doi: 10.1042/bj2750171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Briggs G. E., Haldane J. B. A Note on the Kinetics of Enzyme Action. Biochem J. 1925;19(2):338–339. doi: 10.1042/bj0190338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bruns C. M., Hubatsch I., Ridderström M., Mannervik B., Tainer J. A. Human glutathione transferase A4-4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products. J Mol Biol. 1999 May 7;288(3):427–439. doi: 10.1006/jmbi.1999.2697. [DOI] [PubMed] [Google Scholar]
  9. Caccuri A. M., Ascenzi P., Antonini G., Parker M. W., Oakley A. J., Chiessi E., Nuccetelli M., Battistoni A., Bellizia A., Ricci G. Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase. J Biol Chem. 1996 Jul 5;271(27):16193–16198. doi: 10.1074/jbc.271.27.16193. [DOI] [PubMed] [Google Scholar]
  10. Cameron A. D., Sinning I., L'Hermite G., Olin B., Board P. G., Mannervik B., Jones T. A. Structural analysis of human alpha-class glutathione transferase A1-1 in the apo-form and in complexes with ethacrynic acid and its glutathione conjugate. Structure. 1995 Jul 15;3(7):717–727. doi: 10.1016/s0969-2126(01)00206-4. [DOI] [PubMed] [Google Scholar]
  11. Dirr H., Reinemer P., Huber R. Refined crystal structure of porcine class Pi glutathione S-transferase (pGST P1-1) at 2.1 A resolution. J Mol Biol. 1994 Oct 14;243(1):72–92. doi: 10.1006/jmbi.1994.1631. [DOI] [PubMed] [Google Scholar]
  12. Dirr H., Reinemer P., Huber R. X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. Eur J Biochem. 1994 Mar 15;220(3):645–661. doi: 10.1111/j.1432-1033.1994.tb18666.x. [DOI] [PubMed] [Google Scholar]
  13. García-Sáez I., Párraga A., Phillips M. F., Mantle T. J., Coll M. Molecular structure at 1.8 A of mouse liver class pi glutathione S-transferase complexed with S-(p-nitrobenzyl)glutathione and other inhibitors. J Mol Biol. 1994 Apr 1;237(3):298–314. doi: 10.1006/jmbi.1994.1232. [DOI] [PubMed] [Google Scholar]
  14. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  15. Habig W. H., Pabst M. J., Jakoby W. B. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974 Nov 25;249(22):7130–7139. [PubMed] [Google Scholar]
  16. Hayes J. D., Pulford D. J. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol. 1995;30(6):445–600. doi: 10.3109/10409239509083491. [DOI] [PubMed] [Google Scholar]
  17. Ji X., Zhang P., Armstrong R. N., Gilliland G. L. The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution. Biochemistry. 1992 Oct 27;31(42):10169–10184. doi: 10.1021/bi00157a004. [DOI] [PubMed] [Google Scholar]
  18. Ji X., von Rosenvinge E. C., Johnson W. W., Tomarev S. I., Piatigorsky J., Armstrong R. N., Gilliland G. L. Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods. Biochemistry. 1995 Apr 25;34(16):5317–5328. doi: 10.1021/bi00016a003. [DOI] [PubMed] [Google Scholar]
  19. Kolm R. H., Sroga G. E., Mannervik B. Participation of the phenolic hydroxyl group of Tyr-8 in the catalytic mechanism of human glutathione transferase P1-1. Biochem J. 1992 Jul 15;285(Pt 2):537–540. doi: 10.1042/bj2850537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lian L. Y. NMR structural studies of glutathione S-transferase. Cell Mol Life Sci. 1998 Apr;54(4):359–362. doi: 10.1007/s000180050164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liu S., Zhang P., Ji X., Johnson W. W., Gilliland G. L., Armstrong R. N. Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione S-transferase. J Biol Chem. 1992 Mar 5;267(7):4296–4299. [PubMed] [Google Scholar]
  23. Mannervik B., Awasthi Y. C., Board P. G., Hayes J. D., Di Ilio C., Ketterer B., Listowsky I., Morgenstern R., Muramatsu M., Pearson W. R. Nomenclature for human glutathione transferases. Biochem J. 1992 Feb 15;282(Pt 1):305–306. doi: 10.1042/bj2820305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mannervik B. The isoenzymes of glutathione transferase. Adv Enzymol Relat Areas Mol Biol. 1985;57:357–417. doi: 10.1002/9780470123034.ch5. [DOI] [PubMed] [Google Scholar]
  25. McDonagh P. D., Judah D. J., Hayes J. D., Lian L. Y., Neal G. E., Wolf C. R., Roberts G. C. Determinants of specificity for aflatoxin B1-8,9-epoxide in alpha-class glutathione S-transferases. Biochem J. 1999 Apr 1;339(Pt 1):95–101. [PMC free article] [PubMed] [Google Scholar]
  26. McTigue M. A., Williams D. R., Tainer J. A. Crystal structures of a schistosomal drug and vaccine target: glutathione S-transferase from Schistosoma japonica and its complex with the leading antischistosomal drug praziquantel. J Mol Biol. 1995 Feb 10;246(1):21–27. doi: 10.1006/jmbi.1994.0061. [DOI] [PubMed] [Google Scholar]
  27. Nieslanik B. S., Dabrowski M. J., Lyon R. P., Atkins W. M. Stopped-flow kinetic analysis of the ligand-induced coil-helix transition in glutathione S-transferase A1-1: evidence for a persistent denatured state. Biochemistry. 1999 May 25;38(21):6971–6980. doi: 10.1021/bi9829130. [DOI] [PubMed] [Google Scholar]
  28. Principato G. B., Danielson U. H., Mannervik B. Relaxed thiol substrate specificity of glutathione transferase effected by a non-substrate glutathione derivative. FEBS Lett. 1988 Apr 11;231(1):155–158. doi: 10.1016/0014-5793(88)80722-1. [DOI] [PubMed] [Google Scholar]
  29. Raghunathan S., Chandross R. J., Kretsinger R. H., Allison T. J., Penington C. J., Rule G. S. Crystal structure of human class mu glutathione transferase GSTM2-2. Effects of lattice packing on conformational heterogeneity. J Mol Biol. 1994 May 20;238(5):815–832. doi: 10.1006/jmbi.1994.1336. [DOI] [PubMed] [Google Scholar]
  30. Reinemer P., Dirr H. W., Ladenstein R., Schäffer J., Gallay O., Huber R. The three-dimensional structure of class pi glutathione S-transferase in complex with glutathione sulfonate at 2.3 A resolution. EMBO J. 1991 Aug;10(8):1997–2005. doi: 10.1002/j.1460-2075.1991.tb07729.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ricci G., Caccuri A. M., Lo Bello M., Rosato N., Mei G., Nicotra M., Chiessi E., Mazzetti A. P., Federici G. Structural flexibility modulates the activity of human glutathione transferase P1-1. Role of helix 2 flexibility in the catalytic mechanism. J Biol Chem. 1996 Jul 5;271(27):16187–16192. doi: 10.1074/jbc.271.27.16187. [DOI] [PubMed] [Google Scholar]
  32. Simula T. P., Glancey M. J., Wolf C. R. Human glutathione S-transferase-expressing Salmonella typhimurium tester strains to study the activation/detoxification of mutagenic compounds: studies with halogenated compounds, aromatic amines and aflatoxin B1. Carcinogenesis. 1993 Jul;14(7):1371–1376. doi: 10.1093/carcin/14.7.1371. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Sluis-Cremer N., Naidoo N. N., Kaplan W. H., Manoharan T. H., Fahl W. E., Dirr H. W. Determination of a binding site for a non-substrate ligand in mammalian cytosolic glutathione S-transferases by means of fluorescence-resonance energy transfer. Eur J Biochem. 1996 Oct 15;241(2):484–488. doi: 10.1111/j.1432-1033.1996.00484.x. [DOI] [PubMed] [Google Scholar]
  35. Stenberg G., Board P. G., Mannervik B. Mutation of an evolutionarily conserved tyrosine residue in the active site of a human class Alpha glutathione transferase. FEBS Lett. 1991 Nov 18;293(1-2):153–155. doi: 10.1016/0014-5793(91)81174-7. [DOI] [PubMed] [Google Scholar]
  36. Widersten M., Björnestedt R., Mannervik B. Involvement of the carboxyl groups of glutathione in the catalytic mechanism of human glutathione transferase A1-1. Biochemistry. 1996 Jun 18;35(24):7731–7742. doi: 10.1021/bi9601619. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Wilce M. C., Parker M. W. Structure and function of glutathione S-transferases. Biochim Biophys Acta. 1994 Mar 16;1205(1):1–18. doi: 10.1016/0167-4838(94)90086-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES