Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1997 Mar;113(3):779–786. doi: 10.1104/pp.113.3.779

Purification and Structural and Kinetic Characterization of the Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase from the Crassulacean Acid Metabolism Plant, Pineapple.

KEJ Tripodi 1, F E Podesta 1
PMCID: PMC158196  PMID: 12223643

Abstract

Pyrphosphate-dependent phosphofructokinase (PFP) was purified to electrophoretic homogeneity from illuminated pineapple (Ananas comosus) leaves. The purified enzyme consists of a single subunit of 61.5 kD that is immunologically related to the potato tuber PFP [beta] subunit. The native form of PFP likely consists of a homodimer of 97.2 kD, as determined by gel filtration. PFP's glycolytic activity was strongly dependent on pH, displaying a maximum at pH 7.7 to 7.9. Gluconeogenic activity was relatively constant between pH 6.7 and 8.7. Activation by Fru-2,6-bisphosphate (Fru-2,6-P2) was dependent on assay pH. In the glycolytic direction, it activated about 10-fold at pH 6.7, but only 2-fold at pH 7.7. The gluconeogenic reaction was only weakly affected by Fru-2,6-P2. The true substrates for the PFP forward and reverse reactions were Fru-6-phosphate and Mg-pyrophosphate, and Fru-1,6-P2, orthophosphate, and Mg2+, respectively. The results suggest that pineapple PFP displays regulatory properties consistent with a pH-based regulation of its glycolytic activity, in which a decrease in cytosolic pH caused by nocturnal acidification during Crassulacean acid metabolism, which could curtail its activity, is compensated by a parallel increase in its sensitivity to Fru-2,6-P2. It is also evident that the [beta] subunit alone is sufficient to confer PFP with a high catalytic rate and the regulatory properties associated with activation by Fru-2,6-P2.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Bertagnolli B. L., Cook P. F. Kinetic mechanism of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii. Biochemistry. 1984 Aug 28;23(18):4101–4108. doi: 10.1021/bi00313a014. [DOI] [PubMed] [Google Scholar]
  2. Bertagnolli B. L., Younathan E. S., Voll R. J., Cook P. F. Kinetic studies on the activation of pyrophosphate-dependent phosphofructokinase from mung bean by fructose 2,6-bisphosphate and related compounds. Biochemistry. 1986 Aug 12;25(16):4682–4687. doi: 10.1021/bi00364a034. [DOI] [PubMed] [Google Scholar]
  3. Botha A. M., Botha F. C. Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits. Plant Physiol. 1991 Aug;96(4):1185–1192. doi: 10.1104/pp.96.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brooks S. P. A simple computer program with statistical tests for the analysis of enzyme kinetics. Biotechniques. 1992 Dec;13(6):906–911. [PubMed] [Google Scholar]
  5. Carlisle S. M., Blakeley S. D., Hemmingsen S. M., Trevanion S. J., Hiyoshi T., Kruger N. J., Dennis D. T. Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the alpha- and beta-subunits and with the ATP-dependent phosphofructokinase. J Biol Chem. 1990 Oct 25;265(30):18366–18371. [PubMed] [Google Scholar]
  6. Carnal N. W., Black C. C. Phosphofructokinase activities in photosynthetic organisms : the occurrence of pyrophosphate-dependent 6-phosphofructokinase in plants and algae. Plant Physiol. 1983 Jan;71(1):150–155. doi: 10.1104/pp.71.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carnal N. W., Black C. C. Pyrophosphate-dependent 6-phosphofructokinase, a new glycolytic enzyme in pineapple leaves. Biochem Biophys Res Commun. 1979 Jan 15;86(1):20–26. doi: 10.1016/0006-291x(79)90376-0. [DOI] [PubMed] [Google Scholar]
  8. Carnal N. W., Black C. C. Soluble Sugars as the Carbohydrate Reserve for CAM in Pineapple Leaves : Implications for the Role of Pyrophosphate:6-Phosphofructokinase in Glycolysis. Plant Physiol. 1989 May;90(1):91–100. doi: 10.1104/pp.90.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng H. F., Tao M. Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase. J Biol Chem. 1990 Feb 5;265(4):2173–2177. [PubMed] [Google Scholar]
  10. Christopher J. T., Holtum JAM. Patterns of Carbon Partitioning in Leaves of Crassulacean Acid Metabolism Species during Deacidification. Plant Physiol. 1996 Sep;112(1):393–399. doi: 10.1104/pp.112.1.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Duff S. M., Moorhead G. B., Lefebvre D. D., Plaxton W. C. Phosphate Starvation Inducible ;Bypasses' of Adenylate and Phosphate Dependent Glycolytic Enzymes in Brassica nigra Suspension Cells. Plant Physiol. 1989 Aug;90(4):1275–1278. doi: 10.1104/pp.90.4.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fahrendorf T., Holtum J. A., Mukherjee U., Latzko E. Fructose 2,6-bisphosphate, carbohydrate partitioning, and crassulacean Acid metabolism. Plant Physiol. 1987 May;84(1):182–187. doi: 10.1104/pp.84.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kruger N. J., Dennis D. T. Molecular properties of pyrophosphate:fructose-6-phosphate phosphotransferase from potato tuber. Arch Biochem Biophys. 1987 Jul;256(1):273–279. doi: 10.1016/0003-9861(87)90446-2. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Montavon P., Kruger N. J. Substrate specificity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase from potato tuber. Plant Physiol. 1992 Aug;99(4):1487–1492. doi: 10.1104/pp.99.4.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moorhead G. B., Plaxton W. C. High-yield purification of potato tuber pyrophosphate: fructose-6-phosphate 1-phosphotransferase. Protein Expr Purif. 1991 Feb;2(1):29–33. doi: 10.1016/1046-5928(91)90005-4. [DOI] [PubMed] [Google Scholar]
  17. Nielsen T. H. Fructose-1,6-Bisphosphate Is an Allosteric Activator of Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase. Plant Physiol. 1995 May;108(1):69–73. doi: 10.1104/pp.108.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Penefsky H. S. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1977 May 10;252(9):2891–2899. [PubMed] [Google Scholar]
  19. Phillips N. F., Li Z. Kinetic mechanism of pyrophosphate-dependent phosphofructokinase from Giardia lamblia. Mol Biochem Parasitol. 1995 Jul;73(1-2):43–51. doi: 10.1016/0166-6851(95)00087-h. [DOI] [PubMed] [Google Scholar]
  20. Plaxton W. C. Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf. Eur J Biochem. 1989 May 1;181(2):443–451. doi: 10.1111/j.1432-1033.1989.tb14745.x. [DOI] [PubMed] [Google Scholar]
  21. Plaxton William C. THE ORGANIZATION AND REGULATION OF PLANT GLYCOLYSIS. Annu Rev Plant Physiol Plant Mol Biol. 1996 Jun;47(NaN):185–214. doi: 10.1146/annurev.arplant.47.1.185. [DOI] [PubMed] [Google Scholar]
  22. Podestá F. E., Moorhead G. B., Plaxton W. C. Potato tuber pyrophosphate-dependent phosphofructokinase: effect of thiols and polyalcohols on its intrinsic fluorescence, oligomeric structure, and activity in dilute solutions. Arch Biochem Biophys. 1994 Aug 15;313(1):50–57. doi: 10.1006/abbi.1994.1357. [DOI] [PubMed] [Google Scholar]
  23. Podestá F. E., Plaxton W. C. Plant cytosolic pyruvate kinase: a kinetic study. Biochim Biophys Acta. 1992 Nov 20;1160(2):213–220. doi: 10.1016/0167-4838(92)90010-b. [DOI] [PubMed] [Google Scholar]
  24. Yan T. F., Tao M. Multiple forms of pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase from wheat seedlings. Regulation by fructose 2,6-bisphosphate. J Biol Chem. 1984 Apr 25;259(8):5087–5092. [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES