title: 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 [gamma-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-Km (0.1 microM) 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 (Ki = 5 microM) 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-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), 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 Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. 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 (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.
authors.affiliation: Laboratoire de Chimie Physiologique, Université Catholique de Louvain, Brussels, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: François
authors.forename: E
authors.initials: E
authors.lastname: Van Schaftigen
authors.forename: H G
authors.initials: HG
authors.lastname: Hers
mesh_terms: D002384:Catalysis; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000378:metabolism*; D006598:Hexosediphosphates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007201:Indicator Dilution Techniques; D007700:Kinetics; D025481:Phosphofructokinase-2; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification*; D011494:Protein Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism
chemical_list: D005635:Fructosediphosphates; D006598:Hexosediphosphates; C027652:fructose 2,6-diphosphate; D010770:Phosphotransferases; D011494:Protein Kinases; D025481:Phosphofructokinase-2