The key role of fructose-2,6-bis-phosphate in the temporal organization of carbohydrate metabolism. An auto-oscillating mathematical model

A mathematical model describing the periodical temporal organization of the open futile cycle fructose-6-phosphate in equilibrium fructose-1,6-bisphosphate (F6P in equilibrium F1,6P2) is investigated. The oscillations in this cycle are caused by the regulatory cycle F6P in equilibrium fructise-2,6-bisphosphate (F2,6P2), catalyzed by phosphofructokinase-2 (PFK-2) with a cascade of covalent chemical modification. The apparent product activation of PFK-2 by F2,6P2 together with the F2,6P2 outflux from the regulatory cycle create square-shaped oscillations in the concentration of F2,6P2, a powerful reciprocal regulator of the enzymes of the futile cycle F6P in equilibrium F1,6P2. Compared to the mechanisms of the autonomous regulation of the F6P in equilibrium F1,6P2 cycle suggested previously, the new one provides an excellent temporal separation of the glycolytic and gluconeogenic pathways and possesses a considerably larger region of existence of the self-oscillatory behaviour.     

Phosphofructo-2-kinase/fructose-2,6-bisphosphatase modulates oscillations of pancreatic islet metabolism

Pulses of insulin from pancreatic beta-cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. It has been proposed that a positive feedback circuit exists within the glycolytic pathway, the autocatalytic activation of phosphofructokinase-1 (PFK1), which endows pancreatic beta-cells with the ability to generate oscillations in metabolism. Flux through PFK1 is controlled by the bifunctional enzyme PFK2/FBPase2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) in two ways: via (1) production/degradation of fructose-2,6-bisphosphate (Fru2,6-BP), a potent allosteric activator of PFK1, as well as (2) direct activation of glucokinase due to a protein-protein interaction. In this study, we used a combination of live-cell imaging and mathematical modeling to examine the effects of inducibly-expressed PFK2/FBPase2 mutants on glucose-induced Ca(2+) pulsatility in mouse islets. Irrespective of the ability to bind glucokinase, mutants of PFK2/FBPase2 that increased the kinase:phosphatase ratio reduced the period and amplitude of Ca(2+) oscillations. Mutants which reduced the kinase:phosphatase ratio had the opposite effect. These results indicate that the main effect of the bifunctional enzyme on islet pulsatility is due to Fru2,6-BP alteration of the threshold for autocatalytic activation of PFK1 by Fru1,6-BP. Using computational models based on PFK1-generated islet oscillations, we then illustrated how moderate elevation of Fru-2,6-BP can increase the frequency of glycolytic oscillations while reducing their amplitude, with sufficiently high activation resulting in termination of slow oscillations. The concordance we observed between PFK2/FBPase2-induced modulation of islet oscillations and the models of PFK1-driven oscillations furthermore suggests that metabolic oscillations, like those found in yeast and skeletal muscle, are shaped early in glycolysis.     

Fructose 2,6-bisphosphate and glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity due to autocatalytic activation by fructose-1,6-P2. Fructose-2,6-P2 is an even more potent activator of phosphofructokinase and is competitive with fructose-1,6-P2 in binding and kinetic studies. The possible role and effects of fructose-2,6-P2 on the oscillating system were therefore examined. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, fructose-2,6-P2 slowly accumulated to 50 nM in 1 h. The nearly monotonic rise, in contrast to the 50-fold oscillations in fructose-1,6-P2, indicated no involvement of fructose-2,6-P2 in the oscillatory process. Addition of 0.5 microM fructose-2,6-P2 blocked the oscillations, and there was negligible appearance of glycolytic intermediates from fructose-1,6-P2 to phosphoenolpyruvate, although similar amounts of lactate accumulated. In the presence of 0.2 microM fructose-2,6-P2, there were small, transient accumulations of fructose-1,6-P2, suggesting aborted activations of phosphofructokinase. Oscillations were not blocked by 0.1 microM fructose-2,6-P2. The average [ATP]/[ADP] ratio in the presence of 0.2 or 0.5 microM fructose-2,6-P2 was half the value in its absence, demonstrating the advantage of the oscillatory behavior in maintaining a high energy state. In the presence of higher, near physiological levels of ATP and citrate, inhibitors which reduce the affinity of phosphofructokinase for fructose-2,6-P2, glycolytic oscillations were not blocked by 1 microM fructose-2,6-P2, its approximate concentration in vivo.     

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.     

Covalent control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: insights into autoregulation of a bifunctional enzyme.

The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.     

Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12

Summary We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.Abbreviations G1P glucose-1-phosphate - G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1-6DP fructose-1,6-diphosphate - PEP phosphoenol pyruvate     

Rat hepatoma (HTC) cell 6-phosphofructo-2-kinase differs from that in liver and can be separated from fructose-2,6-bisphosphatase

6-Phosphofructo-2-kinase was purified from rat liver and hepatoma (HTC) cells. The HTC cell enzyme had kinetic properties different from those of the liver enzyme (more sensitive to inhibition by citrate and not inhibited by sn-glycerol 3-phosphate) and was not a substrate of the cyclic-AMP-dependent protein kinase. Unlike the liver enzyme, which is bifunctional and phosphorylated by fructose 2,6-[2-32P]bisphosphate, the HTC cell enzyme contained no detectable fructose-2,6-bisphosphatase activity and phosphorylation by fructose 2,6-[2-32P]-bisphosphate could not be detected. HTC cell fructose-2,6-bisphosphatase could be separated from 6-phosphofructo-2-kinase activity by purification. Antibodies raised against liver 6-phosphofructo-2-kinase did not precipitate HTC cell fructose-2,6-bisphosphatase whose kinetic properties were completely different from those of the liver enzyme.     

Analysis of allosteric mechanisms of suppressing parasitic recirculation in the futile cycle fructose-6-P--fructose-1,6-P2

The uncontrollable substrate recirculation in the central futile cycle (FC) in the carbohydrate energy metabolism fructose-6-P (F6P) in equilibrium or formed from fructose-1,6-P2 (FBP), makes it impossible to maintain a stable level of ATP because of its wasteful expenditure in the cycle reactions which are equivalent to the ATPase reaction and also because of the diversion of FBP from glycolytic phosphorylation of ADP. It follows from the analysis of a mathematical model of the carbohydrate energy metabolism that the allosteric inhibition of fructosebisphosphatase (FBPase) by FBP and AMP leads to suppression of the recirculation in the FC and recovery of the ability of glycolysis to stabilize the level of ATP with high accuracy. The allosteric activation of phosphofrucktokinase (PFK) by AMP couples the expenditure of ATP and F6P in the FC with ATP consumption by a load.     

High resolution phosphorus NMR spectroscopy of transfer ribonucleic acids

Summary A new activator of phosphofructokinase, which is bound to the enzyme and released during its purification, has been discovered. Its structure has been determined as -D Fructose-2,6-P₂ by chemical synthesis, analysis of various degradation products and NMR. D-Fructose-2,6-P₂ is the most potent activator of phosphofructokinase and relieves inhibition of the enzyme by ATP and citrate. It lowers the K_m for fructose-6-P from 6 mM to 0.1 mM.Fructose-6-P,2-kinase catalyzes the synthesis of fructose-2,6-P₂ from fructose-6-P and ATP, and the enzyme has been partially purified. The degradation of fructose-2,6-P₂ is catalyzed by fructose-2,6-bisphosphatase. Thus a metabolic cycle could occur between fructose-6-P and fructose-2,6-P₂, which are catalyzed by these two opposing enzymes. The activities of these enzymes can be controlled by phosphorylation. Fructose-6-P,2-kinase is inactivated by phosphorylation catalyzed by either cAMP dependent protein kinase or phosphorylase kinase. The inactive, phospho-fructose-6-P,2-kinase is activated by dephosphorylation catalyzed by phosphorylase phosphatase. On the other hand, fructose-2,6-bisphosphatase is activated by phosphorylation catalyzed by cAMP dependent protein kinase.Investigation into the hormonal regulation of phosphofructokinase reveals that glucagon stimulates phosphorylation of phosphofructokinase which results in decreased affinity for fructose-2,6-P₂, and decreases the fructose-2,6-P₂ levels. This decreased level in fructose-2,6-P₂ appears to be due to the decreased synthesis by inactivation of fructose-2,6-P₂,2-kinase and increased degradation as a result of activation of fructose-2,6-bisphosphatase. Such a reciprocal change in these two enzymes has been demonstrated in the hepatocytes treated by glucagon and epinephrine. The implications of these observations in respect to possible coordinated controls of glycolysis and glycogen metabolism are discussed.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification,kinetics and immunological properties

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mol · l^-1). The inhibition was counteracted by inorganic phosphate and, particularly, by glycerol 3-phosphate. In the presence of inorganic phosphate and glycerol 3-phosphate the frog muscle fructose-2,6-bisphosphatase was much more sensitive to fructose 6-phosphate inhibition than was the rat M-type fructose-2,6-bisphosphatase. No change in kinetics and no phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was observed after incubation with protein kinase C and a Ca²⁺/calmodulin-dependent protein kinase. The kinetics of frog muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, although they would favour an initial increase in fructose 2,6-bisphosphate in exercising frog muscle, cannot fully account for the changes in fructose 2,6-bisphosphate observed in muscle of exercising frog. Regulatory mechanisms not yet studied must be involved in working frog muscle in vivo.Abbreviations BSA bovine serum albumin - Ca/CAMK Ca²⁺/calmodulin-dependent protein kinase (EC 2.7.1.37) - CL anti-l-type PFK-21 FBPase-2 antiserum - DTT dithiothreitol - EP phosphorylated enzyme intermediate - FBPase-2 fructose-2,6-bisphosphatase (EC 3.1.3.46) - F2,6P₂ fructose 2,6-bisphosphate - I_0,5 inhibitor concentration required to decrease enzyme activity by 50% - MCL-2 anti-PFK-2/FBPase-2 antiserum - M_r relative molecular mass - PEG polyethylene glycol - PFK-1 6-phosphofructo-1-kinase (EC 2.7.1.11) - PKF-2 6-phosphofructo-2-kinase (EC 2.7.1.105) - PKA protein kinase A = cyclic AMP-dependent protein kinase (EC 2.7.1.37) - PKC protein kinase C (EC 2.7.1.37) - SDS sodium dodecylsulphate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - U unit of enzyme activity     

Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library. Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner

The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSAXXVAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5'-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion.     

Isolation and characterization of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver

6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were copurified to homogeneity from bovine liver. The purification scheme consisted of polyethylene glycol precipitation, anion-exchange and Blue-Sepharose chromatography, substrate elution from phosphocellulose, and gel filtration. The bifunctional enzyme had an apparent molecular weight of 102,000 and consisted of two subunits (Mr 49,000). The kinase had a Km for ATP of 12 microM and a S0.5 for fructose 6-phosphate of 150 microM while the bisphosphatase had a Km for fructose 2,6-bisphosphate of 7 microM. Both activities were subject to modulation by various effectors. Inorganic phosphate stimulated both activities, while alpha-glycerolphosphate inhibited the kinase and stimulated the bisphosphatase. The pH optimum for the 6-phosphofructo-2-kinase activity was 8.5, while the fructose-2,6-bisphosphatase reaction was maximal at pH 6.5. Incubation of the purified enzyme with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in 32P incorporation to the extent of 0.7 mol/mol enzyme subunit with concomitant inhibition of the kinase activity and activation of the bisphosphatase activity. The mediation of the bisphosphatase reaction by a phosphoenzyme intermediate was suggested by the isolation of a stable labeled phosphoenzyme when the enzyme was incubated with fructose 2,6-[2-32P]bisphosphate. The pH dependence of hydrolysis of the phospho group suggested that it was linked to the N3 of a histidyl residue. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver has properties essentially identical to those of the rat liver enzyme, suggesting that hepatic fructose 2,6-bisphosphate metabolism is under the same control in both species.     

ADP modulates the dynamic behavior of the glycolytic pathway of Escherichia coli

A mathematical model that includes biochemical interactions among the PTS system, phosphofructokinase (PFK), and pyruvate kinase (PK) is used to evaluate the dynamic behavior of the glycolytic pathway of Escherichia coli under steady-state conditions. The influence of ADP, phosphoenolpyruvate (PEP), and fructose-6-phosphate (F6P) on the dynamic regulation of this pathway is also analyzed. The model shows that the dynamic behavior of the system is affected significantly depending on whether ADP, PEP, or F6P is considered constant a steady state. Sustained oscillations are observed only when dADP/dt not equal 0 and completely suppressed if dADP/dt = 0 at any steady-state value. However, when PEP or F6P is constant, the system evolves toward the formation of stable limit cycles with periods ranging from 0.2 min to hours.     

Contribution of different protein phosphatases to the dephosphorylation of 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat liver.

The nature of rat liver protein phosphatases involved in the dephosphorylation of the glycolytic key enzyme 6-phosphofructo-1-kinase and the regulatory enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was investigated. In terms of the classification system proposed by Ingebritsen & Cohen [(1983) Eur. J. Biochem. 132, 255-261], only the type-2 protein phosphatases 2A (which can be separated into 2A1 and 2A2) and 2C act on these substrates. Fractionation of rat liver extracts by anion-exchange chromatography and gel filtration revealed that protein phosphatase 2A is responsible for most of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase phosphatase activity (activity ratio 2A/2C = 4:1). On the other hand, 6-phosphofructo-1-kinase phosphatase activity is equally distributed between protein phosphatases 2A (2A1 plus 2A2) and 2C. In addition, the possible role of low-Mr compounds for the control of purified protein phosphatase 2C was examined. At near-physiological concentrations, none of the metabolites studied significantly affected the rate of dephosphorylation of 6-phosphofructo-1-kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, pyruvate kinase or fructose-1,6-bisphosphatase.     

Carbon partitioning in leaves and tubers of transgenic potato plants with reduced activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase

The role of fructose-2,6-bisphosphate (Fru-2,6-P₂) in regulation of carbon metabolism was investigated in transgenic potato plants ( Solanum tuberosum L. cv Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC 2.7.1.105)/fructose-2,6-bisphosphatase (F26BPase, EC 3.1.3.46) in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P₂ was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic ¹⁴CO₂ metabolism, showed that in plant lines with reduced Fru-2,6-P₂ level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P₂ only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-¹⁴C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-¹⁴C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P₂ is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P₂ on tuber metabolism was limited.     

Identification of a nerve ending-enriched 29-kDa protein,labeled with [3-32P]1,3-bisphosphoglycerate,as monophosphoglycerate mutase: inhibition by fructose-2,6-bisphosphate via enhancement of dephosphorylation

Glucose metabolism is of vital importance in normal brain function. Evidence indicates that glycolysis, in addition to production of ATP, plays an important role in maintaining normal synaptic function. In an effort to understand the potential involvement of a glycolytic intermediate(s) in synaptic function, we have prepared [3-32P]1,3-bisphosphoglycerate and [32P]3-phosphoglycerate and sought their interaction with a specific nerve-ending protein. We have found that a 29-kDa protein is the major component labeled with either [3-32P]1,3-bisphosphoglycerate or [32P]3-phosphoglycerate. The protein was identified as monophosphoglycerate mutase (PGAM). This labeling was remarkably high in the brain and synaptosomal cytosol fraction, consistent with the importance of glycolysis in synaptic function. Of interest, fructose-2,6-bisphosphate (Fru-2,6-P2) inhibited PGAM phosphorylation and enzyme activity. Moreover, Fru-2,6-P2 potently stimulated release of [32P]phosphate from the 32P-labeled PGAM (EC50 = 1 microM), suggesting that apparent reduction of PGAM phosphorylation and enzyme activity by Fru-2,6-P2 may be due to stimulation of dephosphorylation of PGAM. The significance of these findings is discussed.     

Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Biol. Chem. 260, 13995-14002). However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity. Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme. The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme. However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme. Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant. In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase. The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme. The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme. The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6-phospho group of fructose 6-phosphate/fructose 2,6-bisphosphate; 2) the fructose 6-phosphate binding site is responsible for substrate inhibition; 3) Inorganic phosphate activates fructose-2,6-bisphosphatase by competing with fructose 6-phosphate for the same site; and 4) Lys-356 is not involved in 6-phosphofructo-2-kinase substrate/product binding or catalysis.     

Upregulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in strawberry

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) is a cytosolic enzyme catalyzing the first committed step in glycolysis by reversibly phosphorylating fructose-6-phosphate to fructose-1,6-bisphosphate. The position of PFP in glycolytic and gluconeogenic metabolism, as well as activity patterns in ripening strawberry, suggest that the enzyme may influence carbohydrate allocation to sugars and organic acids. Fructose-2,6-bisphosphate activates and tightly regulates PFP activity in plants and has hampered attempts to increase PFP activity through overexpression. Heterologous expression of a homodimeric isoform from Giardia lamblia, not regulated by fructose-2,6-bisphosphate, was therefore employed to ensure in vivo increases in PFP activity. The coding sequence was placed into a constitutive expression cassette under control of the cauliflower mosaic virus 35S promoter and introduced into strawberry by Agrobacterium tumefaciens-mediated transformation. Heterologous expression of PFP resulted in an up to eightfold increase in total activity in ripe berries collected over two consecutive growing seasons. Total sugar and organic acid content of transgenic berries harvested during the first season were not affected when compared to the wild type, however, fructose content increased at the expense of sucrose. In the second season, total sugar content and composition remained unchanged while the citrate content increased slightly. Considering that PFP catalyses a reversible reaction, PFP activity appears to shift between gluconeogenic and glycolytic metabolism, depending on the metabolic status of the cell.