Effect of Buffer Solutions on Activation of Shamouti Orange Pyrophosphate-Dependent Phosphofructokinase by Fructose 2,6-bisphosphate

IUBMB Life. 2000 Feb;49(2):149-52. doi: 10.1080/15216540050022485.

Abstract

Shamouti phosphofructokinase (PFP) activation depends on the presence of fructose 2,6-bisphosphate (Fru-2,6-P2) in the glycolytic reaction. The effect of activation by Fru-2,6-P2 differs considerably, however, according to the buffer (pH 8.0) in which the reaction is performed: Ka = 2.77 +/- 0.3 nM in Hepes-NaOH and 7.75 +/- 1.49 nM in Tris-HCl. The presence of chloride ions (39 mM) in the Tris-HCl buffer inhibits PFP. Indeed, when using a Hepes-NaOH buffer and then adding 39 mM NaCl, Ka = 8.12 +/- 0.52 nM. The Ki for chloride ions is approximately 21.7 mM. In the gluconeogenic reaction, Shamouti PFP generally showed a high endogenous activity. Addition of Fru-2,6-P2 did not modify the velocity and the Vmax of the enzyme; however, its presence increased the affinity of the enzyme for Fru-1,6-P2 from 200 +/- 15.6 microM in absence of Fru-2,6-P2 to 89 +/- 10.3 microM in its presence (10 microM). In the presence of chloride (39 mM), the affinity for the substrate decreased with K(m) = 150 +/- 14 microM. The calculated Ki for chloride ions equals 56.9 mM. In both the glycolytic and the gluconeogenic reactions, Vmax is not affected; therefore, the inhibition mode of chloride is competitive.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Buffers
  • Citrus / enzymology*
  • Enzyme Activation
  • Fructosediphosphates / pharmacology*
  • Gluconeogenesis
  • Glycolysis
  • Kinetics
  • Phosphotransferases / metabolism*
  • Solutions
  • Substrate Specificity

Substances

  • Buffers
  • Fructosediphosphates
  • Solutions
  • fructose 2,6-diphosphate
  • Phosphotransferases
  • pyrophosphate-fructose 6-phosphate 1-phosphotransferase