User menu

Accès à distance ? S'identifier sur le proxy UCLouvain

Glucosephosphate isomerase from Trypanosoma brucei. Cloning and characterization of the gene and analysis of the enzyme.

Primary tabs

Marchand, Maurice [UCL] Kooystra, U Wierenga, R K Lambeir, A M Van Beeumen, J Opperdoes, Frederik [UCL] Michels, Paulus [UCL]

In Trypanosoma brucei the enzyme glucose-6-phosphate isomerase, like most other enzymes of the glycolytic pathway, resides in a microbody-like organelle, the glycosome. Here we report a detailed study of this enzyme, involving a determination of its kinetic properties and the cloning and sequence analysis of its gene. The gene codes for a polypeptide of 606 amino acids, with a calculated Mr of 67280. The protein predicted from the gene sequence has 54-58% positional identity with its yeast and mammalian counterparts. Compared to those other glucose-6-phosphate isomerases the trypanosomal enzyme contains an additional 38-49 amino acids in its N-terminal domain, as well as a number of small insertions and deletions. The additional amino acids are responsible for the 5-kDa-larger subunit mass of the T. brucei enzyme, as measured by gel electrophoresis. The glucose-6-phosphate isomerase of the trypanosome has no excess of positive residues and, consequently, no high isoelectric point, in contrast to the other glycolytic enzymes that are present in the glycosome. However, similar to other glycosomal proteins analyzed so far, specific clusters of positive residues can be recognized in the primary structure. Comparison of the kinetic properties of the T. brucei glucose-6-phosphate isomerase with those of the yeast and rabbit muscle enzymes did not reveal major differences. The three enzymes have very similar pH profiles. The affinity for the substrate fructose 6-phosphate (Km = 0.122 mM) and the inhibition constant for the competitive inhibitor gluconate 6-phosphate (Ki = 0.14 mM) are in the same range as those of the similar enzymes. The Km shows the same strong dependence on salt as the rabbit muscle enzyme, although somewhat less than the yeast glucose-6-phosphate isomerase. The trypanocidal drug suramin inhibits the T. brucei and yeast enzymes to the same extent (Ki = 0.29 and 0.36 mM, respectively), but it had no effect on the rabbit muscle enzyme. Agaricic acid, a potent inhibitor of various glycosomal enzymes of T. brucei, has also a strong, irreversible effect on glucose-6-phosphate isomerase, while leaving the yeast and mammalian enzymes relatively unaffected.

Document type Article de périodique (Journal article) – Comparative Study, Journal Article, Research Support, Non-U.S. Gov't
Access type Accès restreint
Publication date 1989
Journal information "European journal of biochemistry / FEBS" - Vol. 184, no. 2, p. 455-64 (1989)
issn 0014-2956
Publication status Publié
Affiliation UCL - MD/BICL - Département de biochimie et de biologie cellulaire
MESH Subject Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA - genetics - isolation & purification ; Genes ; Glucose-6-Phosphate Isomerase - genetics - metabolism ; Humans ; Molecular Sequence Data ; Restriction Mapping ; Sequence Homology, Nucleic Acid ; Trypanosoma brucei brucei - enzymology - genetics
Links
  1. Opperdoes Fred R., Borst Piet, Localization of nine glycolytic enzymes in a microbody-like organelle inTrypanosoma brucei: The glycosome, 10.1016/0014-5793(77)80476-6
  2. Opperdoes F., Compartmentation Of Carbohydrate Metabolism In Trypanosomes, 10.1146/annurev.micro.41.1.127
  3. Misset O., Eur. J. Biochem., 144, 475 (1984)
  4. Misset O., Eur. J. Biochem., 157, 441 (1986)
  5. Osinga K. A., EMBO J., 4, 3811 (1985)
  6. Michels P. A. M., EMBO J., 5, 1049 (1986)
  7. Swinkels B. W., EMBO J., 5, 1291 (1986)
  8. MARCHAND M, POLISZCZAK A, GIBSON W, WIERENGA R, OPPERDOES F, MICHELS P, Characterization of the genes for fructose-bisphosphate aldolase in Trypanosoma brucei, 10.1016/0166-6851(88)90121-1
  9. Wierenga R. K., EMBO J., 6, 215 (1987)
  10. Hart D. T., EMBO J., 6, 1403 (1987)
  11. Swinkels B. W., EMBO J., 7, 1159 (1988)
  12. Calder William A, Parr C.R, Karl D.P, Energy content of eggs of the brown kiwi Apteryx australis; an extreme in avian evolution, 10.1016/0300-9629(78)90226-8
  13. Widmer Giovanni, Dvorak James A., Miles Michael A., A biochemical comparison of glucosephosphate isomerase isozymes from Trypanosoma cruzi, 10.1016/0167-4838(86)90197-4
  14. 14. E. A. Noltman, and P. D. Boyer (1972 ) inThe enzymes () vol. 6 , pp.272 -354 , Academic Press, New York.
  15. Achari A., Phil. Trans. R. Soc. Lond. B, 293, 145 (1981)
  16. Palmieri R. H., J. Biol. Chem., 257, 7965 (1982)
  17. Gibson D. R., J. Biol. Chem., 255, 9369 (1980)
  18. Tekamp-Olson P., Gene, 73, 153 (1988)
  19. Chaput Mariella, Claes Victor, Portetelle Daniel, Cludts Isabelle, Cravador Alfredo, Burny Arsène, Gras Hélène, Tartar André, The neurotrophic factor neuroleukin is 90% homologous with phosphohexose isomerase, 10.1038/332454a0
  20. Faik P., Nature, 332, 455 (1988)
  21. Gurney M. E., Science, 234, 566 (1986)
  22. Shaw P. J., J. Mol. Biol., 109, 475 (1977)
  23. Opperdoes F.R., Aarsen P.N., van der Meer C., Borst P., Trypanosoma brucei: An evaluation of salicylhydroxamic acid as a trypanocidal drug, 10.1016/0014-4894(76)90082-5
  24. Bernards André, Lex H.T., Van der Ploeg A., Frasch Carlos C., Borst Piet, Boothroyd John C., Coleman Sharon, Cross George A.M., Activation of trypanosome surface glycoprotein genes involves a duplication-transposition leading to an altered 3′ end, 10.1016/0092-8674(81)90391-3
  25. Frischauf A., J. Mol. Biol., 170, 827 (1983)
  26. Aguilera Andr�s, Zimmermann Friedrich K., Isolation and molecular analysis of the phosphoglucose isomerase structural gene of Saccharomyces cerevisiae, 10.1007/bf00330521
  27. 27. T. Maniatis, E. F. Fritsch, and J. Sambrook (1982 ) Molecular cloning: a laboratory manual , p.545 , Cold Spring Harbor Laboratory Press, New York.
  28. Yanisch-Perron Celeste, Vieira Jeffrey, Messing Joachim, Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors, 10.1016/0378-1119(85)90120-9
  29. Hanahan D., J. Mol. Biol., 166, 557 (1983)
  30. Wahl G. M., Proc. Natl Acad. Sci. USA, 76, 3683 (1979)
  31. Johnson P. J., Gene, 43, 213 (1986)
  32. Ploeg L. H. T., Cell, 37, 77 (1984)
  33. Rigby Peter W.J., Dieckmann Marianne, Rhodes Carl, Berg Paul, Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I, 10.1016/0022-2836(77)90052-3
  34. Smith H. O., Methods Enzymol., 65, 371 (1980)
  35. Sanger F., Proc. Natl Acad. Sci. USA, 74, 5463 (1977)
  36. Sanger F., Coulson A.R., Barrell B.G., Smith A.J.H., Roe B.A., Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing, 10.1016/0022-2836(80)90196-5
  37. Messing Joachim, [2] New M13 vectors for cloning, Recombinant DNA Part C (1983) ISBN:9780121820015 p.20-78, 10.1016/0076-6879(83)01005-8
  38. Gracy R. W., Methods Enzymol., 41, 392 (1973)
  39. Stein S., Arch. Biochem. Biophys., 155, 203 (1973)
  40. Gibson W. C., Mol. Biochem. Parasitol., 16, 231 (1985)
  41. Gibson Wendy C., Borst Piet, Size-fractionation of the small chromosomes of Trypanozoon and Nannomonas trypanosomes by pulsed field gradient gel electrophoresis, 10.1016/0166-6851(86)90033-2
  42. Kyte J., J. Mol. Biol., 157, 105 (1982)
  43. Hines M. C., Biochemistry, 2, 770 (1963)
  44. Schray K. J., Arch. Biochem. Biophys., 192, 241 (1979)
  45. Lu H. S., Arch. Biochem. Biophys., 212, 347 (1981)
  46. Lu H. S., J. Biol. Chem., 256, 785 (1981)
  47. Pullan L. M., Arch. Biochem. Biophys., 221, 489 (1983)
  48. Bacchi C. J., J. Bacteriol., 98, 23 (1969)
  49. Michels P. A. M., EMBO J., 3, 1345 (1984)
  50. Ploeg L. H. T., Cell, 39, 213 (1984)
  51. Tait A., Nature, 287, 536 (1980)
  52. Hart David T., Opperdoes Fred R., The occurrence of glycosomes (microbodies) in the promastigote stage of four major Leishmania species, 10.1016/0166-6851(84)90110-5
  53. Opperdoes F. R., Parasitology, 82, 10 (1981)
  54. Blackburn M. N., J. Biol. Chem., 247, 1170 (1972)
  55. Dyson J. E. D., J. Biol. Chem., 243, 1401 (1968)
  56. O'Connell E. L., J. Biol. Chem., 248, 2225 (1973)
  57. 57. M. E. Gurney (1987 ) Genbank, release 57, accession no. K03515.
Bibliographic reference Marchand, Maurice ; Kooystra, U ; Wierenga, R K ; Lambeir, A M ; Van Beeumen, J ; et. al. Glucosephosphate isomerase from Trypanosoma brucei. Cloning and characterization of the gene and analysis of the enzyme.. In: European journal of biochemistry / FEBS, Vol. 184, no. 2, p. 455-64 (1989)
Permanent URL http://hdl.handle.net/2078.1/27027