A role of phosphofructokinase in pH-dependent regulation of glycolysis

Abstract

1. 1. Glycolysis in a cell-free extract from rat diaphragm was stimulated at elevated pH in the presence of higher concentrations of ATP. A similar situation was also observed in the case of intact-cell preparations of rat diaphragm. 2. 2. It was found that the rate of inhibition of phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) activity by excess ATP was strictly dependent upon the pH; at pH 7.3 an increase in ATP concentration from 1 to 1.7 mM resulted in a sudden inhibition of phosphofructokinase activity, whereas at pH 7.6 such an effect was not observed until the ATP level was raised to 2–3 mM (0.05 mM fructose 6-phosphate was employed as substrate). Consequently phosphofructokinase activity was profoundly influenced by quite a minute change in pH when larger amounts of ATP were present in the glycolytic system. 3. 3. Since the glucose 6-phosphate which accumulated during phosphofructokinase inhibition was inhibitory to hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the pH-induced stimulation of phosphofructokinase caused an exaggerated acceleration of overall glycolysis. 4. 4. Preincubation of the cell-free extract without substrates (aging) rendered phosphofructokinase protein unresponsive both to the stimulation on raising pH and to the inhibition by ATP. It is tentatively proposed that the affinity of the inhibitory site(s) of phosphofructokinase for ATP is affected by hydrogen ion concentration and that aging causes a change of conformation of the enzyme, thus blocking the control mechanism of phosphofructokinase.