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

Authors

  • Jean FRANÇOIS,

    1. Laboratoire de Chimie Physiologique, Université Catholique de Louvain and International Institute of Cellular and Molecular Pathology
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  • Emile VAN SCHAFTINGEN,

    1. Laboratoire de Chimie Physiologique, Université Catholique de Louvain and International Institute of Cellular and Molecular Pathology
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  • Henri-Géry HERS

    Corresponding author
    1. Laboratoire de Chimie Physiologique, Université Catholique de Louvain and International Institute of Cellular and Molecular Pathology
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  • Note added in proof (received November 9, 1987). After the present manuscript had been sent for publication, Kretchmer et al. (Biochem. J. 246, 755–759, 1987) described the purification of PFK 2 and of the low-Km, FBPase 2 from baker's yeast and found, as we did, that the two enzymes can be separated from each other. In contrast to our results, however, the activity of their low-Km, FBPase 2 was almost equal to that of PFK 2 and could be decreased 2.5-fold upon incubation in the presence of ATP-Mg and the C subunit of the bovine heart cyclic AMP-dependent protein kinase. Using commercial baker's yeast (Levure Royale, Brussels) as a source of enzyme, we have been unable to repeat the observation by Kretchmer et al., but instead obtained results very similar to those reported in this paper. We have no explanation to offer for this discrepancy.

Correspondence to H.-G. Hers, Laboratoire de Chimie Physiologique, UCL-7539, 75 avenue Hippocrate, B-1200 Brussels, Belgium

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-Km, (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 (Ki= 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-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (∼ 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 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, > 50 μM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.

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