Effect of buffer solutions on activation of Shamouti orange pyrophosphate-dependent phosphofructokinase by fructose 2,6-bisphosphate

Shamouti phosphofructokinase (PFP) activation depends on the presence of fructose 2,6-bisphosphate (Fru-2,6-P-2) in the glycolytic reaction. The effect of activation by Fru-2,6-P-2 differs considerably, however, according to the buffer (pH 8.0) in which the reaction is performed: K-a = 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, K-a = 8.12 +/- 0.52 nM, The Ki for chloride ions is similar to 21.7 mM. In the gluconeogenic reaction, Shamouti PFP generally showed a high endogenous activity. Addition of Fru-2,6-P-2 did not modify the velocity and the V-max of the enzyme; however, its presence increased the affinity of the enzyme for Fru-1,6-P-2 from 200 +/- 15.6 mu M in absence of Fru-2,6-P-2 to 89 +/- 10.3 mu M in its presence (10 mu M). In the presence of chloride (39 mM), the affinity for the substrate decreased with K-m = 150 +/- 14 mu M, The calculated K-i for chloride ions equals 56.9 mM, In both the glycolytic and the gluconeogenic reactions, V-max is not affected; therefore, the inhibition mode of chloride is competitive.