Kinetic effects of fructose-1,6-bisphosphate on yeast phosphofructokinase.

Yeast phosphofructokinase is known to be effectively activated by fructose-2,6-bisphosphate and AMP. In the absence of the two effectors, fructose-1,6-bisphosphate activates or inhibits the enzyme according to the concentrations of the substrates and of inorganic phosphate. At cellular concentrations of the substrates, however, the effects of fructose-1,6-bisphosphate are negligible. Whereas the activation of the enzyme by AMP is not affected by fructose-1,6-bisphosphate, the latter was found to diminish strongly the activity of the fructose-2,6-bisphosphate-activated enzyme. Inorganic phosphate amplifies the activating effect of fructose-2,6-bisphosphate and augments also the deactivation of the fructose-2,6-bisphosphate-activated enzyme. The deactivating action of fructose-1,6-bisphosphate with respect to fructose-2,6-bisphosphate dominates at low concentrations of fructose-6-phosphate and high levels of ATP and might be of regulatory significance.

Effects of fructose 1,6-bisphosphate on the activation of yeast phosphofructokinase by fructose 2,6-bisphosphate and AMP.

Fructose 1,6-bisphosphate decreases the activation of yeast 6-phosphofructokinase (ATP:fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) by fructose 2,6-bisphosphate, especially at cellular substrate concentrations. AMP activation of the enzyme is not influenced by fructose 1,6-bisphosphate. Inorganic phosphate increases the activation by fructose 2,6-bisphosphate and augments the deactivation of the fructose 2,6-bisphosphate activated enzyme by fructose 1,6-bisphosphate. Because various states of yeast glucose metabolism differ in the levels of the two fructose bisphosphates, the observed interactions might be of regulatory significance.

Inorganic phosphate amplifies the effects of AMP and fructose-2,6-bisphosphate on yeast phosphofructokinase.

Inorganic phosphate is an important regulator of yeast phosphofructokinase activity. In the absence of AMP and fructose-2,6-bisphosphate the dependence of enzyme activity on the concentration of inorganic phosphate is sigmoidal. AMP and fructose-2,6-bisphosphate increase the affinity of phosphofructokinase to inorganic phosphate. At low fructose-6-phosphate concentrations inorganic phosphate amplifies the activating effect of AMP and fructose-2,6-bisphosphate. Yeast phosphofructokinase is more sensitive to ATP inhibition in the absence of inorganic phosphate than in its presence. While in the absence of inorganic phosphate a definite ATP inhibition prevails even at high levels of AMP or fructose-2,6-bisphosphate, the ATP inhibition can be relieved by the cooperation of inorganic phosphate and fructose-2,6-bisphosphate. These effects of inorganic phosphate provide an explanation for the stimulation of glycolysis under anaerobic conditions by inorganic phosphate at unchanged concentrations of AMP and fructose-2,6-bisphosphate (Lagunas and Gancedo, Eur. J. Biochem. 137, 479-483 (1983)).

Influence of inorganic phosphate on the kinetic properties of yeast phosphofructokinase.

Yeast phosphofructokinase is effectively activated by inorganic phosphate. In the absence of other allosteric stimulators, inorganic phosphate increases the maximum activity of the enzyme only. In the presence of the activators AMP and fructose 2,6-bisphosphate inorganic phosphate causes changes in the maximum activity and the enzyme affinity to fructose 6-phosphate. Inorganic phosphate augments the sensitivity of phosphofructokinase to the activators AMP and fructose 2,6-bisphosphate and increases the respective maximum activities. The extent of activation of the enzyme by inorganic phosphate prevails at low levels of fructose 6-phosphate and high ATP concentrations.