Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Apr 1;363(Pt 1):189–193. doi: 10.1042/0264-6021:3630189

Glutamic acid-65 is an essential residue for catalysis in Proteus mirabilis glutathione S-transferase B1-1.

Nerino Allocati 1, Michele Masulli 1, Enrico Casalone 1, Silvia Santucci 1, Bartolo Favaloro 1, Michael W Parker 1, Carmine Di Ilio 1
PMCID: PMC1222466  PMID: 11903062

Abstract

The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu(65), Ser(103) and Glu(104) are in hydrogen-bonding distance of the N-terminal amino group of the gamma-glutamyl moiety of the co-substrate, GSH. Glu(65) was mutated to either aspartic acid or leucine, and Ser(103) and Glu(104) were both mutated to alanine. Glu(65) mutants (Glu(65)-->Asp and Glu(65)-->Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser(103)-->Ala and Glu(104)-->Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser(103) and Glu(104) are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu(65) is crucial for catalysis.

Full Text

The Full Text of this article is available as a PDF (138.7 KB).

Selected References

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

  1. Allocati N., Casalone E., Masulli M., Ceccarelli I., Carletti E., Parker M. W., Di Ilio C. Functional analysis of the evolutionarily conserved proline 53 residue in Proteus mirabilis glutathione transferase B1-1. FEBS Lett. 1999 Feb 26;445(2-3):347–350. doi: 10.1016/s0014-5793(99)00147-7. [DOI] [PubMed] [Google Scholar]
  2. Allocati N., Casalone E., Masulli M., Polekhina G., Rossjohn J., Parker M. W., Di Ilio C. Evaluation of the role of two conserved active-site residues in beta class glutathione S-transferases. Biochem J. 2000 Oct 15;351(Pt 2):341–346. [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  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. 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]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Casalone E., Allocati N., Ceccarelli I., Masulli M., Rossjohn J., Parker M. W., Di Ilio C. Site-directed mutagenesis of the Proteus mirabilis glutathione transferase B1-1 G-site. FEBS Lett. 1998 Feb 20;423(2):122–124. doi: 10.1016/s0014-5793(98)00080-5. [DOI] [PubMed] [Google Scholar]
  8. Di Ilio C., Aceto A., Piccolomini R., Allocati N., Faraone A., Cellini L., Ravagnan G., Federici G. Purification and characterization of three forms of glutathione transferase from Proteus mirabilis. Biochem J. 1988 Nov 1;255(3):971–975. doi: 10.1042/bj2550971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Graminski G. F., Kubo Y., Armstrong R. N. Spectroscopic and kinetic evidence for the thiolate anion of glutathione at the active site of glutathione S-transferase. Biochemistry. 1989 Apr 18;28(8):3562–3568. doi: 10.1021/bi00434a062. [DOI] [PubMed] [Google Scholar]
  11. Habig W. H., Jakoby W. B. Assays for differentiation of glutathione S-transferases. Methods Enzymol. 1981;77:398–405. doi: 10.1016/s0076-6879(81)77053-8. [DOI] [PubMed] [Google Scholar]
  12. Hayes J. D., McLellan L. I. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res. 1999 Oct;31(4):273–300. doi: 10.1080/10715769900300851. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Jakobsson P. J., Morgenstern R., Mancini J., Ford-Hutchinson A., Persson B. Common structural features of MAPEG -- a widespread superfamily of membrane associated proteins with highly divergent functions in eicosanoid and glutathione metabolism. Protein Sci. 1999 Mar;8(3):689–692. doi: 10.1110/ps.8.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Mannervik B., Danielson U. H. Glutathione transferases--structure and catalytic activity. CRC Crit Rev Biochem. 1988;23(3):283–337. doi: 10.3109/10409238809088226. [DOI] [PubMed] [Google Scholar]
  19. Manoharan T. H., Gulick A. M., Reinemer P., Dirr H. W., Huber R., Fahl W. E. Mutational substitution of residues implicated by crystal structure in binding the substrate glutathione to human glutathione S-transferase pi. J Mol Biol. 1992 Jul 20;226(2):319–322. doi: 10.1016/0022-2836(92)90949-k. [DOI] [PubMed] [Google Scholar]
  20. Mignogna G., Allocati N., Aceto A., Piccolomini R., Di Ilio C., Barra D., Martini F. The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes. Eur J Biochem. 1993 Feb 1;211(3):421–425. doi: 10.1111/j.1432-1033.1993.tb17566.x. [DOI] [PubMed] [Google Scholar]
  21. Nishida M., Harada S., Noguchi S., Satow Y., Inoue H., Takahashi K. Three-dimensional structure of Escherichia coli glutathione S-transferase complexed with glutathione sulfonate: catalytic roles of Cys10 and His106. J Mol Biol. 1998 Aug 7;281(1):135–147. doi: 10.1006/jmbi.1998.1927. [DOI] [PubMed] [Google Scholar]
  22. Perito B., Allocati N., Casalone E., Masulli M., Dragani B., Polsinelli M., Aceto A., Di Ilio C. Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis. Biochem J. 1996 Aug 15;318(Pt 1):157–162. doi: 10.1042/bj3180157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reinemer P., Prade L., Hof P., Neuefeind T., Huber R., Zettl R., Palme K., Schell J., Koelln I., Bartunik H. D. Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 A resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. J Mol Biol. 1996 Jan 19;255(2):289–309. doi: 10.1006/jmbi.1996.0024. [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. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [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. Widersten M., Kolm R. H., Björnestedt R., Mannervik B. Contribution of five amino acid residues in the glutathione-binding site to the function of human glutathione transferase P1-1. Biochem J. 1992 Jul 15;285(Pt 2):377–381. doi: 10.1042/bj2850377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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