Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6‐bisphosphate in yeast

Abstract

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [γ-³²P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of ³²P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-K_m, (0.1 μM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-³²P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (K_i= 5 μM) and stimulated 2–3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-K_m fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-K_m (∼ 100 μM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and P_i, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or P_i. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (K_m, > 50 μM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.