Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1977 Sep 1;165(3):525–537. doi: 10.1042/bj1650525

Effect of substrates and effectors on the reversible inactivation of pig spleen phosphofructokinase by adenosine triphosphate

Maurice J Weidemann 1,*, Marysia E Kolbuch-Braddon 1, Peter E Hickman 1,†
PMCID: PMC1164936  PMID: 21659

Abstract

1. To investigate the mechanism of the reversible inactivation of pig spleen phosphofructokinase by ATP, the effect of order of addition of reactants (substrates, effectors and enzyme solution) was studied by preincubating the enzyme before assay with various combinations of its substrates and effectors. 2. Preincubation of the enzyme with MgATP or ATP at pH7.0 before addition of fructose 6-phosphate caused a rapid and much greater inhibition of activity than that observed when the reaction (carried out at identical substrate concentrations) was initiated with enzyme. 3. The rapid inhibition caused by preincubation with ATP, together with the sigmoidal response to fructose 6-phosphate and activation by AMP, were all blocked by prior photo-oxidation of the enzyme with Methylene Blue, which selectively destroys the inhibitory binding site for ATP [Ahlfors & Mansour (1969) J. Biol. Chem. 244, 1247–1251]. 4. Fructose 6-phosphate, but not Mg2+, protected phosphofructokinase from inhibition during preincubation with ATP in a manner that was sigmoidally dependent on the fructose 6-phosphate concentration. 5. Mg2+, by protecting the enzyme from the inhibitory effect of preincubation at low pH (7.0) and by preventing its activation during preincubation with fructose 6-phosphate, demonstrated both a weak activating effect in the absence of the other substrates and a stronger inhibitory effect in the presence of fructose 6-phosphate. 6. Positive effectors (K+, NH4+, AMP and aspartate) protected the enzyme from inhibition during preincubation with MgATP in proportion to their potency as activators, but citrate potentiated the ATP inhibition. Pi significantly slowed the inactivation process without itself acting as a positive effector. 7. The non-linear dependence of the initial rate of the unmodified enzyme on protein concentration (associated with increased positive homotropic co-operativity to fructose 6-phosphate) was intensified by preincubation with ATP and abolished by photo-oxidation. 8. The results are interpreted in terms of an association–dissociation model which postulates that protonation, at low pH, of a photo-oxidation-sensitive inhibitory site for ATP allows more rapid dissociation of an active tetramer to an inactive dimeric species.

Full text

PDF
525

Selected References

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

  1. Achilles W., Cumme G. A., Hoppe H. Acid dissociation and complex formation of glycolytic metabolites with magnesium, potassium and sodium. Determination of acidity and stability constants by pH titration. Acta Biol Med Ger. 1973;31(6):763–770. [PubMed] [Google Scholar]
  2. Ahlfors C. E., Mansour T. E. Studies on heart phosphofructokinase. Desensitization of the enzyme to adenosine triphosphate inhibition. J Biol Chem. 1969 Mar 10;244(5):1247–1251. [PubMed] [Google Scholar]
  3. Colombo G., Tate P. W., Girotti A. W., Kemp R. G. Interaction of inhibitors with muscle phosphofructokinase. J Biol Chem. 1975 Dec 25;250(24):9404–9412. [PubMed] [Google Scholar]
  4. Emerk K., Frieden C. Rabbit muscle phosphofructokinase: the effect of the state of the enzyme and assay procedure on the kinetic properties. Arch Biochem Biophys. 1975 May;168(1):210–218. doi: 10.1016/0003-9861(75)90243-x. [DOI] [PubMed] [Google Scholar]
  5. Frieden C. Kinetic aspects of regulation of metabolic processes. The hysteretic enzyme concept. J Biol Chem. 1970 Nov 10;245(21):5788–5799. [PubMed] [Google Scholar]
  6. Hickman P. E., Weidemann M. J. Modulation of phosphofructokinase activity by positive effectors. FEBS Lett. 1973 Dec 15;38(1):1–3. doi: 10.1016/0014-5793(73)80497-1. [DOI] [PubMed] [Google Scholar]
  7. Hickman P. E., Weidemann M. J. The purification and properties of pig spleen phosphofructokinase. Biochem J. 1975 Nov;151(2):327–336. doi: 10.1042/bj1510327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hill D. E., Hammes G. G. An equilibrium binding study of the interaction of fructose 6-phosphate and fructose 1,6-bisphosphate with rabbit muscle phosphofructokinase. Biochemistry. 1975 Jan 28;14(2):203–213. doi: 10.1021/bi00673a003. [DOI] [PubMed] [Google Scholar]
  9. Hulme E. C., Tipton K. F. The dependence of phosphofructokinase kinetics upon protein concentration. FEBS Lett. 1971 Jan 25;12(4):197–200. doi: 10.1016/0014-5793(71)80019-4. [DOI] [PubMed] [Google Scholar]
  10. Kemp R. G. Allosteric properties of muscle phosphofructokinase. I. Binding of magnesium adenosine triphosphate to the inhibitory site. Biochemistry. 1969 Aug;8(8):3162–3168. doi: 10.1021/bi00836a005. [DOI] [PubMed] [Google Scholar]
  11. Kemp R. G., Krebs E. G. Binding of metabolites by phosphofructokinase. Biochemistry. 1967 Feb;6(2):423–434. doi: 10.1021/bi00854a009. [DOI] [PubMed] [Google Scholar]
  12. Kemp R. G. Rabbit liver phosphofructokinase. Comparison of some properties with those of muscle phosphofructokinase. J Biol Chem. 1971 Jan 10;246(1):245–252. [PubMed] [Google Scholar]
  13. Kühn B., Jacobasch G., Gerth C., Rapoport S. M. Kinetic properties of the phosphofructokinase from erythrocytes of rats and rabbits. 2. The influence of effectors under nearly cellular conditions. Eur J Biochem. 1974 Apr 16;43(3):443–450. doi: 10.1111/j.1432-1033.1974.tb03430.x. [DOI] [PubMed] [Google Scholar]
  14. Lee L. M., Krupka R. M., Cook R. A. Cooperativity in human erythrocyte phosphofructokinase. Biochemistry. 1973 Aug 28;12(18):3503–3508. doi: 10.1021/bi00742a024. [DOI] [PubMed] [Google Scholar]
  15. Lorenson M. Y., Mansour T. E. Studies on heart phosphofructokinase. Binding properties of native enzyme and of enzyme desensitized to allosteric control. J Biol Chem. 1969 Dec 10;244(23):6420–6431. [PubMed] [Google Scholar]
  16. Lowry O. H., Passonneau J. V. Kinetic evidence for multiple binding sites on phosphofructokinase. J Biol Chem. 1966 May 25;241(10):2268–2279. [PubMed] [Google Scholar]
  17. MONOD J., WYMAN J., CHANGEUX J. P. ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. J Mol Biol. 1965 May;12:88–118. doi: 10.1016/s0022-2836(65)80285-6. [DOI] [PubMed] [Google Scholar]
  18. Mansour T. E. Phosphofructokinase. Curr Top Cell Regul. 1972;5:1–46. [PubMed] [Google Scholar]
  19. Massey T. H., Deal W. C., Jr Unusual, metabolite-dependent solubility properties of phosphofructokinase. The basis for a new and rapid purification from liver, kidney, and other tissues. J Biol Chem. 1973 Jan 10;248(1):56–62. [PubMed] [Google Scholar]
  20. Ramaiah A., Tejwani G. A. Interconvertible forms of phosphofructokinase of rabbit liver. Biochem Biophys Res Commun. 1970;39(6):1149–1156. doi: 10.1016/0006-291x(70)90680-7. [DOI] [PubMed] [Google Scholar]
  21. Tejwani G. A., Kaur J., Ananthanarayanan M., Ramaiah A. Concentrations of various effectors and substrates of phosphofructokinase in the jejunum of rat and their relation to the lack of Pasteur effect in this tissue. Biochim Biophys Acta. 1974 Nov 25;370(1):120–129. doi: 10.1016/0005-2744(74)90038-2. [DOI] [PubMed] [Google Scholar]
  22. Wakid N., Mansour T. E. Factors influencing the stability of heart phosphofructokinase. Mol Pharmacol. 1965 Jul;1(1):53–59. [PubMed] [Google Scholar]
  23. Wenzel K. W., Kurganov B. I., Zimmermann G., Yakovlev V. A., Schellenberger W., Hofmann E. Self-association of human erythrocyte phosphofructokinase. Kinetic behaviour in dependence on enzyme concentration and mode of association. Eur J Biochem. 1976 Jan 2;61(1):181–190. doi: 10.1111/j.1432-1033.1976.tb10010.x. [DOI] [PubMed] [Google Scholar]
  24. Zimmermann G., Wenzel K. W., Gauer J., Hofmann E. Studies on the association behaviour of human-erythrocyte phosphofructokinase. Eur J Biochem. 1973 Dec 17;40(2):501–505. doi: 10.1111/j.1432-1033.1973.tb03219.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES