Phosphate dependency of phosphofructokinase 2

Abstract

Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate. The concentration of inorganic phosphate that allowed half‐maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8. The effect of phosphate was to increase V and to decrease K_m for fructose 6‐phosphate, without affecting K_m for ATP. Citrate and P‐enolpyruvate inhibited the enzyme non‐competitively with fructose 6‐phosphate and independently of the concentration of inorganic phosphate. Phosphorylation of the enzyme by the catalytic subunit of cyclic‐AMP‐dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate. A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves. By contrast, the fructose 2,6‐bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate. Phosphate increased this activity about threefold when measured in the absence of added fructose 6‐phosphate and a half‐maximal effect was reached at approximately 0.5 mM phosphate. Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6‐bisphosphatase by fructose 6‐phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect.