Fructose 2-phosphate, an intermediate of the dephosphorylation of fructose 2,6-bisphosphate with a purified yeast enzyme

Abstract

A fructose-2,6-bisphosphate dephosphorylating enzyme was 3000-fold purified to electrophoretic homogeneity from Saccharomyces cerevisiae. Half-maximal activity was obtained at pH 6.0 with 6 pM fructose 2,6-bisphosphate and 0.15mM Mg2+. On incubation for 90min with fructose 2,6-bisphosphate, about 80% of the substrate appears with an almost linear time dependence as fructose. In the first 30 min a substance accumulates to about 40% of the consumed fructose 2,6-bisphosphate which forms free fructose on mild acid treatment. Formation of fructose 6-phosphate was negligible. The mild-acid-labile intermediate was identified as fructose 2-phosphate by comparative ion-exchange chromatography with authentic fructose 2-phosphate synthesized from fructose l-phosphate [Pontis, H. . The data suggest the reaction sequence fructose 2,6-bisphosphate + fructose 2-phosphate -+ fructose. The designation fructose-2,6-bisphosphate 6-phosphohydrolase is proposed for the enzyme described here.Fructose 2,6-bisphosphate [Fru(2,6)P2] has' been discovered to be a potent activator of 6-phosphofructokinase-1 and inhibitor of fructose-l,6-bisphosphatase in liver, other mammalian tissues and yeast [l, 21. Fru(2,6)P2 is formed in liver by an enzyme catalyzing ATP-dependent phosphorylation of Fru6P in the 2-position. It has been shown that the same enzyme protein also catalyzes dephosphorylation of Fru(2,6)P2 to Fru6P and inorganic phosphate. Modification of the kinase activity to the phosphatase activity of this enzyme is effected by cyclic-AMP-dependent protein phosphorylation. The reverse modification is the result of dephosphorylation of the enzyme [3, 41. In yeast cells a kinase catalyzing phosphorylation of Fru6P to Fru(2,6)P2 has also been described [5, 61. The kinase is activated by cyclic-AMPdependent phosphorylation [5, 71. This is in contrast to liver, where cyclic-AMP-dependent phosphorylation inactivates fructose-6-phosphate kinase-2 and increases fructose-2,6-bisphosphatase activity. In yeast no activity dephosphorylating Fru(2,6)P2 to Fru6P has been detected [5, 71. It is evident from these results that the metabolism of Fru(2,6)P2 in yeast is different from that in mammalian tissues. In the present paper purification of an enzyme to electrophoretic homogeneity from yeast which dephosphorylates Fru(2,6)P2 is described. Fru2P, which has not been described previously as an enzymatic product, and fructose were shown to be the dephosphorylation products.

Ion-Exchange Chromatography Separates Activities Synthesizing and Degrading Fructose 2,6-Bisphosphate from C3 and C4 Leaves but Not from Rat Liver

“…Our present results do not rule out the possibility that the Fru 6-P,2K from plant leaves is capable of catalyzing the hydrolysis of Fru 2,6-P2 to fructose 2-phosphate and Pi as has been reported for the enzyme from yeast (14). Such an activity, which would not be detected by the spectrophotometric assay currently used, may account for the low Fru 2,6-P2ase activity (Fru 2,6-P2ase bioassay based on Fru 2,6-P2 disappearance) detected by others and reported to co-chromatograph with Fru 6-P,2K (10,16).…”

Fructose 2,6-Bisphosphate Hydrolyzing Enzymes in Higher Plants

“…In the case of Fru-2,6-P2, however, it produces about 10-fold more Fru-2-P than Fru-6-P, indicating that it splits the furanosyl phosphate linkage less easily than the ester phosphate one. The occurrence of an enzyme forming Fru-2-P from Fru-2,6-P2 has not been previously reported in plants, but is well established in yeast (8,17); in the latter case, the enzyme formed similar amounts of Fru-2-P and of Fru-6-P.…”

Signal Transduction Dynamics of the Protein Kinase-A/Phosphofructokinase-2 System in Saccharomyces cerevisiae

“…A second specific phosphatase (K m =6 +M) degrading F-2,6-BP to F2P and inorganic phosphate (Purwin et al, 1987;Francois et al, 1988).…”