Fructose 2,6-bisphosphate and plant carbohydrate metabolism

The control of the fructose 2,6-bisphosphate (Fru2,6P₂) concentration and its possible role in controlling carbohydrate synthesis and degradation are discussed. This regulator metabolite is involved in the fine tuning of photosynthetic metabolism, and in controlling photosynthetic partitioning, and may also be involved in the response to hormones, wounding, and changing water relations. Study of the mechanisms controlling Fru2,6P₂ concentrations could reveal insights into how these responses are mediated. However, the detailed action of Fru2,6P₂ requires more attention, especially in respiratory metabolism where the background information about the compartmentation of metabolism between the plastid and cytosol is still inadequate, and the potential role of pyrophosphate has to be clarified.     

Fructose-2,6-bisphosphate in rat mesenteric lymph nodes

1. The fructose-2,6-bisphosphate (Fru-2,6-P2) content of mesenteric lymph nodes was measured in rats. 2. The effects of Fru-2,6-P2 on the activity of 6-phosphofructo-1-kinase (PFK-1) from rat mesenteric lymph nodes were also studied. 3. The affinity of the enzyme for fructose-6-phosphate was increased by Fru-2,6-P2 whereas the inhibition of the enzyme with high concentrations of ATP was released by Fru-2,6-P2. 4. The activity of lymphocyte PFK-1 was highly stimulated in a simultaneous presence of low concentrations of AMP and Fru-2,6-P2. 5. These results show that rat lymphocyte PFK-1 is highly regulated with Fru-2,6-P2 which means that glycolysis in rat lymphocytes is controlled by Fru-2,6-P2.     

Fructose-1,6-bisphosphate,a regulator of metabolism

Summary Fructose-1, 6-bisphosphate affects the rate of a large variety of enzyme reactions. In some instances its role as a physiologic effector is well documented. In many cases the effects of fructose bishosphate on particular enzymes have been demonstrated in vitro but the link to physiologic conditions has not yet been established. It is the purpose of this paper to summarize the scattered findings in fructose bisphosphate as an effector of enzyme reactions and to draw some conclusions about the role of the compound in metabolic regulation.     

Fructose-2,6-bisphosphate Activates Pyrophosphate: d-Fructose 6-Phosphate 1-Phosphotransferase from Euglena gracilis

When hexavalent chromium (Cr⁶⁺) tolerant Pseudomonas ambigua G-l was cultivated in nutrient broth containing 150 ppm Cr^{6 +}, the Cr⁶⁺ content of the broth rapidly decreased. The Cr⁶⁺ reducing enzyme found in a cell-free extract of P. ambigua G-l required NADH but not NADPH as a hydrogen donor for the reduction of Cr^{6 +}. The specific activities of cell-free extracts of several Cr⁶⁺ sensitive mutants derived from P. ambigua G-l showed decreases to one fourth to one tenth of that of P. ambigua G-l. Glucose protected the Cr⁶⁺ reducing enzyme against inac-tivation on dialysis.     

Fructose-2,6-bisphosphate increases the binding affinity of yeast phosphofructokinase to AMP

At saturating concentrations of AMP four molecules of this ligand are bound per octamer of yeast phosphofructokinase. Fructose-2,6-bisphosphate increases the binding affinity of the enzyme to AMP. This indicates synergistic cooperation of the two allosteric activators in the binding process. The stoichiometry of binding is not altered by fructose-2,6-bisphosphate.     

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.     

Pyrophosphate-dependent sucrose metabolism and its activation by fructose 2,6-bisphosphate in sucrose importing plant tissues

In the presence of pyrophosphate and uridine diphosphate, sucrose was cleaved to form glucose 1-phosphate and fructose with soluble extracts from sucrose importing plant tissues. The glucose 1-phosphate then was converted through glycolysis to triose phosphates in a pyrophosphate-dependent pathway which was activated by fructose 2,6-bisphosphate. Much less activity, less than 5%, was found in sucrose exporting tissue extracts from the same plants. These findings suggest that imported sucrose is metabolized in the cytoplasm of plant tissues by utilizing pyrophosphate and that sucrose metabolism is partially regulated by fructose 2,6-bisphosphate.     

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.     

Ethylene-induced increase in fructose-2,6-bisphosphate in plant storage tissues

Treatment of carrot roots with ethylene led to: (a) a doubling of the fructose-2,6-bisphosphate content; (b) a general increase in the concentration of glycolytic intermediates; and (c) an increase in the extractable activity of fructose-6-phosphate,2-kinase, the enzyme synthesizing fructose-2,6-bisphosphate from fructose-6-phosphate and adenosine triphosphate.     

Rapid oscillations in fructose 2,6-bisphosphate levels in plant tissues

The fructose 2,6-bisphosphate (Fru 2,6-P₂) content of pea, Pisum sativum, roots and leaves were measured following flooding with water and found to change in times of minutes and to exhibit oscillatory-type changes. Each organ changes its Fru 2,6-P₂ content in a unique pattern in response to environmental disturbances such as flooding or light. For example, when roots of intact illuminated pea plants are flooded, roots decrease their Fru 2,6-P₂ content while simultaneously leaves increase their Fru 2,6-P₂ content; but both organs exhibit oscillatory-type patterns within flooding time of about 30 minutes. Half-change times can be as rapid as 2 to 3 minutes. The endogenous extractable activity of the root pyrophosphate-dependent phosphofructokinase also exhibits an oscillatory pattern upon root immersion slightly after Fru 2,6-P₂ changes occur. We postulate from these results that Fru 2,6-P₂ is a primary signal molecule which enables plants to regulate their metabolism to cope with changing environments.     

Fructose-2,6-bisphosphate is lower in copper deficient rat cerebellum despite higher content of phosphorylated AMP-activated protein kinase

Limitation in copper (Cu) leads to pathophysiology in developing brain. Cu deficiency impairs brain mitochondria and results in high brain lactate suggesting augmented anaerobic glycolysis. AMP activated protein kinase (AMPK) is a cellular energy "master-switch" that is thought to augment glycolysis through phosphorylation and activation phosphofructokinase 2 (PFK2) resulting in increases of the glycolytic stimulator fructose-2,6-bisphosphate (F2,6BP). Previously, Cu deficiency has been shown to augment cerebellar AMPK activation. Cerebella of Cu-adequate (Cu+) and Cu-deficient (Cu-) rat pups were assessed to evaluate if AMPK activation in Cu- cerebella functioned to enhance PFK2 activation and increase F2,BP concentration. Higher levels of pAMPK were detected in Cu- cerebella. However, PFK2 activity, mRNA, and protein abundance were not affected by Cu deficiency. Surprisingly, F2,6BP levels were markedly lower in Cu- cerebella. Lower F2,6BP may be due to inhibition of PFK2 by citrate, as citrate concentration was significantly higher in Cu- cerebella. Data suggest AMPK activation in Cu- cerebellum does not augment glycolysis through a PFK2 mechanism. Furthermore, other metabolite data suggest that glycolysis may actually be blunted, since levels of glucose and glucose-6-phosphate were higher in Cu- cerebella than controls.     

Characterization of a Mn-dependent Fructose-1,6-bisphosphate Aldolase in Deinococcus radiodurans

The key enzyme of the glycolytic pathway of Deinococcus radiodurans, fructose-1,6-bisphosphate aldolase, could be induced independently by glucose and Mn. The enzyme exhibited the characteristics of the metal-dependent Class II aldolases. Unlike most Class II aldolases, the deinococcal aldolase preferred Mn, not Zn, as a cofactor. The fbaA gene encoding the deinococcal aldolase was cloned and the protein overproduced in various Escherichia coli expression hosts. However, the overexpressed deinococcal enzyme aggregated and formed inclusion bodies. Dissolving these inclusion bodies by urea and subsequent purification by nickel affinity chromatography, resulted in a protein fraction that exhibited aldolase activity only in the presence of Mn. This active aldolase fraction exhibited masses of about 70 kDa and 35 kDa by gel filtration and by SDS gel electrophoresis, respectively, suggesting that the active aldolase was a dimer.     

Differences in fructose-2,6-bisphosphate metabolism between sections of developing barley leaves

In order to study the regulation of carbohydrate metabolism in leaf tissue the activity of fructose-6-phosphate,2-kinase was determined in individual sections of developing primary leaves of barley. Activity was about 25-fold higher in the leaf tip than in the leaf sheath when measured on a fresh weight basis. There was a gradual increase in enzyme activity from the leaf base to the leaf tip. The higher activity of fructose-6-phosphate,2-kinase in the apical parts of the leaf was associated with higher levels of fructose-2,6-bisphosphate. This was especially pronounced when isolated leaf segments were treated with vanadate and kept in darkness. As compared to the kinase, little difference was observed in the fructose-2,6-bisphospatase activity among leaf sections. The significance of these patterns for regulation of carbohydrate metabolism in different tissues is discussed.     

Effect of benzoate on the metabolism of fructose 2,6-bisphosphate in yeast

When benzoate (2 mM, pH 3.5) was added together with glucose (0.1 M) to a suspension of Saccharomyces cerevisiae in the stationary phase, it caused a relative increase in the concentration of glucose 6-phosphate and fructose 6-phosphate and a decrease in the concentration of fructose 1,6-bisphosphate. These effects are in confirmation of similar observations made by Krebs et al. [Biochem. J. 214, 657-663 (1983)] and are indicative of an inhibition of 6-phosphofructo-1-kinase. Benzoate also caused an about fourfold relative decrease in the concentration of fructose 2,6-bisphosphate, an increase in that of cyclic AMP with no change in that of ATP. It also greatly decreased the activation of 6-phosphofructo-2-kinase, but not that of trehalase, both of which normally occur upon addition of glucose to a yeast suspension. When added 10 min after glucose, benzoate caused a rapid (within 2-3 min) decrease in fructose 2,6-bisphosphate concentration and in 6-phosphofructo-2-kinase activity. In the presence of benzoate, there was also a parallel decrease in the concentration of fructose 2,6-bisphosphate and in the rate of ethanol production when the external pH was dropped from 5.0 to 2.5, with minimal change in the concentration of ATP. Purified 6-phosphofructo-2-kinase was inhibited by benzoate and also by an acid pH. Experiments with cell-free extracts did not provide an explanation for the rapid disappearance of fructose-2,6-bisphosphate or the inactivation of 6-phosphofructo-2-kinase in yeast upon addition of benzoate.