In vitro activation of phosphoglucomutase by fructose 2,6-bisphosphate

The hexose bisphosphate activation of phosphoglucomutase was investigated with both plant (pea and mung bean) and animal (rabbit muscle) sources of the enzyme. Plant phosphoglucomutase was purified about 50-fold from seeds, and to a lesser extent, from seedlings of Pisum sativum L. cv Grenadier and seedlings of Phaseolus aureus. It was found that the plant enzyme was isolated in a mostly dephosphorylated form while commercial rabbit muscle phosphoglucomutase was predominantly in the phosphorylated form. Activation studies were done using the dephosphorylated enzymes. The range of activation constant (K_a) values were obtained for each bisphosphate were: for glucose 1-6-P₂, 0.5 to 1.8; fructose 2,6-P₂, 6 to 11.7; and fructose 1,6-P₂, 7 micromolar, respectively. Fructose 2,6-P₂ is known to occur in both plant and animal tissues at changing levels encompassing the K_a values found in this study; hence, these results implicate fructose 2,6-P₂ as a natural activator of phosphoglucomutase, particularly in plants. Also, glucose 1,6-P₂ has not been found in plants, and the method for measuring glucose 1,6-P₂ by monitoring the activation of phosphoglucomutase is not specific.     

Effects of fructose 2,6-bisphosphate on phosphoglucomutase from plants

Fructose 2,6-bisphosphate affects phosphoglucomutase from plant and animal sources in a similar way. As previously found with rabbit muscle phosphoglucomutase, fructose 2,6-bisphosphate cannot substitute for glucose 1,6-bisphosphate as a cofactor in the reaction catalyzed by phosphoglucomutase from potato tubers, pea seeds, and string-beans. In the presence of glucose 1,6-bisphosphate, fructose 2,6-bisphosphate inhibits phosphoglucomutase from potato tubers. Activation of phosphoglucomutase from plant sources by fructose 2,6-bisphosphate reported by others was probably due to contamination of the commercial preparation of fructose 2,6-bisphosphate by glucose 1,6-bisphosphate.     

Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Rat liver fructose-1,6-bisphosphatase, which was assayed by measuring the release of 32P from fructose 1,6-[1-32P]bisphosphate at pH 7.5, exhibited hyperbolic kinetics with regard to its substrate. beta-D-Fructose 2,6-bisphosphate, an activator of hepatic phosphofructokinase, was found to be a potent inhibitor of the enzyme. The inhibition was competitive in nature and the Ki was estimated to be 0.5 microM. The Hill coefficient for the reaction was 1.0 in the presence and absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate also enhanced inhibition of the enzyme by the allosteric inhibitor AMP. The possible role of fructose 2,6-bisphosphate in the regulation of substrate cycling at the fructose-1,6-bisphosphatase step is discussed.     

Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme. The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations. At a substrate concentration of 20 μM, 50% inhibition was observed at 4.8 μM fructose 2,6-bisphosphate. Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (K_j = 16 μM) and phosphoenolpyruvate caused release of AMP inhibition. However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate. The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate-fructose-1,6-bisphosphatase active site interaction.     

Fructose 2,6-bisphosphate in rat erythrocytes. Inhibition of fructose 2,6-bisphosphate synthesis and measurement by glycerate 2,3-bisphosphate.

The concentration of fructose 2,6-bisphosphate found in freshly isolated erythrocytes was below the limit of detection (20 pmol/ml of packed cells). However, it increased to about 250 pmol/ml of cells when erythrocytes were incubated with glucose at pH 6.9, but not at pH 7.4 or 8.2. This could be explained by variations in the content of glycerate 2,3-bisphosphate, which was found to inhibit 6-phosphofructo-2-kinase, the enzyme responsible for fructose 2,6-bisphosphate synthesis. Glycerate 2,3-bisphosphate was also found to inhibit the potato enzyme (pyrophosphate:fructose-6-phosphate 1-phosphotransferase) used for the measurement of fructose 2,6-bisphosphate.     

Fructose 2,6-bisphosphate inhibition of phosphoglucomutase

Fructose 2,6-bisphosphate inhibits phosphoglucomutase noncompetitively with respect to the cofactor glucose 1,6-bisphosphate. Previous studies from our laboratory had shown that phosphoglucomutase was activated by fructose 2,6-bisphosphate in the absence of added glucose 1,6-bisphosphate. The fructose 2,6-bisphosphate activation previously reported was due to the presence of glucose 1,6-bisphosphate in the commercial preparation of fructose 2,6-bisphosphate.     

Inhibition of fructose bisphosphatase and stimulation of phosphofructokinase by a stable isosteric phosphonate analog of fructose 2,6-bisphosphate

We describe the synthesis of a mixture of D-manno- and D-gluco-2,5-anhydro-1-deoxy-1-phosphonohexitol 6-phosphate via a Horner-Emmons reaction of 2,3,5-tri-O-benzyl-beta-D-arabinofuranose followed by phosphorylation of the equivalent 6-position and subsequent deprotection. This mixture inhibits fructose-1,6-bisphosphatase; the concentration required for half-maximal effect in the presence of 25 microM AMP is approximately 6 microM. The mixture of analogs also stimulates 6-phosphofructo-1-kinase from rabbit liver; the concentration required to reach one-half Vmax was found to be ca. 25 microM at 0.25 mM fructose 6-phosphate and 50 microM AMP. These analogs have replaced the labile anomeric phosphate of fructose 2,6-bisphosphate with a stable methylenephosphonate, and could be of great interest due to their appropriate physiological effects and their chemical stability.     

Evolutionary analysis of fructose 2,6-bisphosphate metabolism

Fructose 2,6-bisphosphate is a potent metabolic regulator in eukaryotic organisms; it affects the activity of key enzymes of the glycolytic and gluconeogenic pathways. The enzymes responsible for its synthesis and hydrolysis, 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) are present in representatives of all major eukaryotic taxa. Results from a bioinformatics analysis of genome databases suggest that very early in evolution, in a common ancestor of all extant eukaryotes, distinct genes encoding PFK-2 and FBPase-2, or related enzymes with broader substrate specificity, fused resulting in a bifunctional enzyme both domains of which had, or later acquired, specificity for fructose 2,6-bisphosphate. Subsequently, in different phylogenetic lineages duplications of the gene of the bifunctional enzyme occurred, allowing the development of distinct isoenzymes for expression in different tissues, at specific developmental stages or under different nutritional conditions. Independently in different lineages of many unicellular eukaryotes one of the domains of the different PFK-2/FBPase-2 isoforms has undergone substitutions of critical catalytic residues, or deletions rendering some enzymes monofunctional. In a considerable number of other unicellular eukaryotes, mainly parasitic organisms, the enzyme seems to have been lost altogether. Besides the catalytic core, the PFK-2/FBPase-2 has often N- and C-terminal extensions which show little sequence conservation. The N-terminal extension in particular can vary considerably in length, and seems to have acquired motifs which, in a lineage-specific manner, may be responsible for regulation of catalytic activities, by phosphorylation or ligand binding, or for mediating protein-protein interactions.     

Effects of fructose 1,6-bisphosphate on the activation of yeast phosphofructokinase by fructose 2,6-bisphosphate and AMP

Fructose 1,6-bisphosphate decreases the activation of yeast 6-phosphofructokinase (ATP:fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) by fructose 2,6-bisphosphate, especially at cellular substrate concentrations. AMP activation of the enzyme is not influenced by fructose 1,6-bisphosphate. Inorganic phosphate increases the activation by fructose 2,6-bisphosphate and augments the deactivation of the fructose 2,6-bisphosphate activated enzyme by fructose 1,6-bisphosphate. Because various states of yeast glucose metabolism differ in the levels of the two fructose bisphosphates, the observed interactions might be of regulatory significance.     

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P₂) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P₂), with K_i for Fru-2,6-P₂ of approximately 0.5 μm. Binding of Fru-2,6-P₂ to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P₂ is enhanced in the presence of a noninhibitory concentration (5 μm) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P_2, unlike the substrate Fru-1,6-P₂, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.     

Activation of muscle phosphofructokinase by fructose 2,6-bisphosphate and fructose 1,6-bisphosphate is differently affected by other regulatory metabolites

Fructose-2,6-P2 and fructose-1,6-P2 are strong activators of muscle phosphofructokinase. They have been shown to be competitive in binding studies, and it is generally thought that they affect the physical and catalytic properties of the enzyme in the same manner. However, there are indications in published data that the effects of the two fructose bisphosphates on phosphofructokinase are not identical. To examine this possibility, the kinetics of activation of rat skeletal muscle phosphofructokinase by the two fructose bisphosphates were compared in the presence of other regulatory metabolites. Citrate greatly increased the K0.5 of the enzyme for fructose-2,6-P2, with little effect on the maximum activation. In contrast, citrate greatly decreased the maximum activation by fructose-1,6-P2, with only a small effect on the K0.5. Changes in the concentrations of the inhibitor ATP or the activator AMP similarly altered the K0.5 for fructose-2,6-P2, but altered the maximum activation by fructose-1,6-P2. Finally, when fructose-1,6-P2 was added in the presence of a given concentration of fructose-2,6-P2, phosphofructokinase activity was decreased if the activation by fructose-2,6-P2 alone was greater than the maximum activation by fructose-1,6-P2 alone. These results are consistent with competition of the two fructose bisphosphates for the same binding site, but indicate that the conformational changes produced by their binding are different.     

Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6-bisphosphate

The activity of pyruvate kinase present in a crude extract of the bloodstream form of Trypanosoma brucei was greatly increased by fructose 2,6-bisphosphate, which converted the saturation curve for phosphoenolpyruvate from a sigmoid into a hyperbola with no change in V. Phosphate and arsenate had an effect opposite to that of fructose 2,6-bisphosphate and the apparent Ka for fructose 2,6-bisphosphate was shifted from 75 nM to 1.5 microM by the presence of 5 mM phosphate. Fructose 1,6-bisphosphate had effects similar to those of fructose 2,6-bisphosphate but at approximately 4000-fold higher concentrations. Pyruvate kinases of Crithidia luciliae and of Leishmania major, two trypanosomatids which are like T. brucei in containing glycosomes, were also stimulated by fructose 2,6-bisphosphate and inhibited by phosphate.     

Inhibition of Rhizopus lactate dehydrogenase by fructose 1,6-bisphosphate

The closely related fungi Rhizopus oryzae and Rhizopus delemar are often used for the production of lactic and fumaric acid, respectively. These organisms differ primarily by their ability to regenerate NAD through alternative fermentative routes. R. oryzae contains an NAD-dependent l-lactate dehydrogenase enzyme, RO-LdhA, that is primarily responsible for production of lactic acid, while both organisms contain another enzyme, LdhB that is thought to be involved in lactic acid production only under certain growth conditions. We have characterized LdhB from both R. oryzae and R. delemar, respectively referred to as RO-LdhB and RD-LdhB in this study, and have determined that RO-LdhB is significantly more effective than RD-LdhB with regard to k_cat/K_m with reductive LDH activity. Only negligible oxidative LDH activity could be measured with both enzymes; however, the presence of an amino terminal fusion with a small ubiquitin-related modifier, SUMO, increased the oxidative activity per μmol protein by more than 100-fold, while having little effect on the reductive LDH activity. We also determined that RO-LdhA, RO-LdhB, and RD-LdhB were all significantly inhibited in a non-competitive manner by fructose 1,6-bisphosphate (FBP) with K_i values of 1.2, 3.2, and 28.8 mM. Intracellular concentrations of FBP were tested with fermentative conditions to demonstrate that this metabolic intermediate does accumulate to levels that would likely cause inhibition of the R. oryzae LDH. Possible reasons for the significant K_i differences between the nearly identical LdhB proteins are discussed.     

A competitive binding assay for fructose 2,6-bisphosphate

A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P2 to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol protomer) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6-[2-32P]P2 and samples containing varying concentrations of fructose 2,6-P2. The resulting stable binary complex is retained on nitrocellulose filters with a binding efficiency of up to 70%. Standard curves obtained with this assay show strict linearity with varying fructose 2,6-P2 in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P2, ATP, and high concentrations of phosphate interfere with this assay. However, the extent of this inhibition is negligible since their tissue contents are one-half to one-tenth that examined. This new assay is simple, direct, rapid, and does not require pretreatment of tissue extracts.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

Regulation of fructose 2,6-bisphosphate levels in Neurospora crassa

Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-bisphosphate at levels of 27 nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-bisphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-bisphosphate. Thus, the above-described regulation of fructose 2,6-bisphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-bisphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-bisphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-bisphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-bisphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.     

Regulation of fructose 2,6-bisphosphate metabolism in human fibroblasts

In the present work, the mechanism involved in the regulation of fructose 2,6-bisphosphate (fructose-2,6-P₂) metabolism in human fibroblasts has been studied. Various agents like serum, insulin and adrenaline known to affect glycolysis have been investigated for their ability to influence fructose 2,6-P₂ metabolism in confluent human fibroblasts. Serum appears to be the most potent activator of fructose-2,6-P₂ levels and capable of inducing a marked increase in 6-phosphofructo-2-kinase (ATP: d-fructose-6-phosphate-2-phosphotransferase), EC 2.7.1. 105). To a lesser extent insulin has the same effects. The increase in enzyme activity elicited by serum and insulin does not require de novo protein synthesis since the process is insensitive to cycloheximide. Incubation of fibroblasts in the presence of adrenaline is responsible for a significant rise in fructose-2,6-P₂ levels without affecting 6-phosphofructo-2-kinase. Similar experiments performed on glucose-starved or cytochalasin B-treated cells show that the effects elicited by all the agents are strictly dependent on glucose availability.     

Regulation of fructose 2,6-bisphosphate concentration in spinach leaves

Fructose-6-phosphate 2-kinase and fructose-2,6-bisphosphatase have been partially purified from spinach leaves and their regulatory properties studied. Fructose-6-phosphate 2-kinase was activated by phosphate and fructose 6-phosphate, and inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fructose-2,6-bisphosphatase was inhibited by fructose 6-phosphate and phosphate. The interaction between these effectors was studied when they were varied, alone or in combination, over a range of concentrations representative of those in the cytosol of spinach leaf cells. In conditions when dihydroxyacetone phosphate or 3-phosphoglycerate rise, as is typical during photosynthesis, the fructose 2,6-bisphosphate level will decrease, which will favour sucrose synthesis. In conditions when fructose 6-phosphate accumulates, fructose 2,6-bisphosphate should rise, which will favour a restriction of sucrose synthesis and promotion of starch synthesis.