The tissue distribution of fructose-2,6-p2 and fructose-6-P,2-kinase in rats and the effect of starvation diabetes and hypoglycemia on hepatic fructose-2,6-P2 and fructose-6-P,2-kinase

The tissue distribution of fructose-2,6-P₂ and fructose-6-P,2-kinase in rats was determined. The highest concentration of fructose-2,6-P₂ was found in liver, followed by brain, heart muscle, kidney, testis and skeletal muscle in decreasing order. Similar results were obtained with fructose-6-P,2-kinase activities in these tissues. Starvation, streptozotocin-induced diabetes or hypoglycemia lowers the fructose-2,6-P₂ levels and fructose-6-P,2-kinase activity in the liver.     

Induction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA by refeeding and insulin

The effects of fasting/refeeding and untreated or insulin-treated diabetes on the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its mRNA in rat liver were determined. Both enzymatic activities fell to 20% of control values with fasting or streptozotocin-induced diabetes and were coordinately restored to normal within 48 h of refeeding or 24 h of insulin administration. These alterations in enzymatic activities were always mirrored by corresponding changes in amount of enzyme as determined by phosphoenzyme formation and immunoblotting. In contrast, mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase did not decrease during starvation or in diabetes, but there was a 3-6-fold increase upon refeeding a high carbohydrate diet to starved rats or insulin treatment of diabetic rats. The decrease of the enzyme in starved or diabetic rats without associated changes in mRNA levels suggests a decrease in the rate of mRNA translation, an increase in enzyme degradation, or both. The rise in enzyme amount and mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with refeeding and insulin treatment suggests an insulin-dependent stimulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression. Northern blots of RNA from heart, brain, kidney, and skeletal muscle probed with restriction fragments of a full-length cDNA from liver showed that only skeletal muscle contained an RNA species that hybridized to any of the probes. Skeletal muscle mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was 2.0 kilobase pairs but in contrast to the liver message (2.2 kilobase pairs) was not regulated by refeeding.     

Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Fructose-6-phosphate,2-kinase:fructose-2,6-bis-phosphatase from rat skeletal muscle has been purified to homogeneity, and its structure and kinetic properties have been determined. The Mr of the native enzyme was 100,000 and the subunit Mr was 54,000. The apparent Km values of fructose-6-P,2-kinase for Fru-6-P and ATP were 56 and 48 microM, respectively. The apparent Km value for Fru-2,6-P2 of fructose-2,6-bis-phosphatase was 0.4 microM, and the Ki for Fru-6-P was 12.5 microM. The enzyme was bifunctional, and the phosphatase activity was 2.5 times higher than the kinase activity. The enzyme was not phosphorylated by cAMP-dependent protein kinase. The amino acid composition of the skeletal muscle enzyme was similar to that of the rat liver enzyme, and the carboxyl terminus sequence (His-Tyr) was the same as that of the liver enzyme. The tryptic peptides generated from the liver and skeletal muscle enzymes were identical except for two peptides. A peptide corresponding to nucleotides 14-28 of the rat liver enzyme was not detected in the skeletal muscle enzyme. A peptide whose amino acid sequence was Thr-Ala-Ser-Ile-Pro-Gln-Phe-Thr-Asn-Ser-Pro-Thr-Met-Val-Ile-Met-Val-Gly-Leu-Pro - Ala-Arg was also isolated. This peptide was the same as that of rat liver enzyme (nucleotides 31-52) containing the phosphorylation site except in the muscle enzyme two amino terminus amino acids, Gly-Ser(P), have been altered to Thr-Ala. Thus, the rat skeletal muscle enzyme is very similar in structure to the rat liver enzyme except for the lack of possibly one peptide and the lack of a phosphorylation site by the substitution of the target Ser with Ala.     

Effect of tolbutamide on fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase in rat liver

The effects of tolbutamide on the activities of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were examined using rat hepatocytes. Tolbutamide stimulated fructose-6-phosphate,2-kinase activity and inhibited fructose-2,6-bisphosphatase activity, resulting in an increase of fructose-2,6-bisphosphate level. Changes in the activities of the enzyme by tolbutamide were due to variation in the Km value, but not dependent on alteration of Vmax. Glucagon inhibition of fructose-2,6-bisphosphate formation resulting from an inactivation of fructose-6-phosphate,2-kinase and an activation of fructose-2,6-bisphosphatase was released by tolbutamide. Tolbutamide stimulation of fructose-2,6-bisphosphate formation through regulation of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase may produce enhancement of glycolysis and inhibition of gluconeogenesis in the liver.     

The mechanism of activation of heart fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Partially purified fructose-6-P,2-kinase:fructose-2,6-bisphosphatase from beef heart was phosphorylated by cAMP protein kinase. The phosphorylated fructose-6-P,2-kinase shows lower Km for Fru-6-P (43 versus 105 microM) and for ATP (0.55 versus 1.3 mM) but no change in the Vmax, compared to those for unphosphorylated enzyme. There was no detectable change in Km or Vmax of fructose-2,6-bisphosphatase activity by the phosphorylation. These changes in heart fructose-6-P,2-kinase were in direct contrast to previous results for the liver isozyme in which phosphorylation led to inhibition of the kinase activity and activation of the phosphatase activity.     

The effect of oxygen on fructose-2, 6-bisphosphate concentration and fructose-6-phosphate, 2-kinase activity in yeast cells

Fructose-2,6-bisphosphate concentration and fructose-6-phosphate,2-kinase activity were measured in yeast cells grown aerobically or anaerobically using glucose as a carbon source. A new improved analytical method using HPLC was employed to measure fructose-2,6-P₂ concentration. Anaerobically-grown yeast cells contain approximately 4-fold higher levels of fructose-2,6-P₂ as compared to aerobically-grown cells in the growth phase of culture. Similarly, fructose-6-P,2-kinase activity is approximately 7-fold higher in the anaerobically-grown cells. These results suggest that the presence of oxygen in the growth medium decreases the content of fructose-2,6-P₂ through inactivation of fructose-6-P,2-kinase.     

Fructose 2,6-bisphosphate and fructose-6-P 2-kinase in Saccharomyces cerevisiae in relation to metabolic state in wild type and fructose-6-P 1-kinase mutant strains

In wild type Saccharomyces cerevisiae, fructose-6-P is known to be in much lower amounts than needed to saturate fructose-6-P 1-kinase in vitro, and the same is true for a mutant with reduced affinity for fructose-6-P, even though its in vivo fructose-6-P concentration is much higher than normal. Both the wild type and mutant fructose-6-P 1-kinases were activated in vitro by fructose-2,6-P2 in the 0.1 microM concentration range, and the effector was present in more than adequate amounts. Hence, it is likely to be necessary for sufficient flux through the fructose-6-P 1-kinase reaction in vivo, and the data also fit with fructose-2,6-P2 acting at different sites on the enzyme from fructose-6-P. In growth on glucose, a variety of wild type strains contained 5-10 microM fructose-2,6-P2, and various fructose-6-P 1-kinase mutant strains had levels of up to 150 microM in the presence of glucose. Fructose-2,6-P2 was also found (0.5-10 microM) in derepressed cultures after glucose exhaustion and in growth on pyruvate. Activities of fructose-6-P 2-kinase in the various strains and situations are also presented. The data generally indicate a correlation between levels of fructose-2,6-P2 and fructose-6-P and suggest that fructose-2,6-P2 is not rapidly degraded after glucose exhaustion.     

Active hepatic glycogen synthesis from gluconeogenic precursors despite high tissue levels of fructose 2,6-bisphosphate

When fasted rats ate regular lab chow there was a lag time of about 2 h before the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2) in liver began to rise from its low basal level. By contrast, in animals refed on a sucrose-based diet hepatic [Fru-2,6-P2] increased 20-fold (to a value of approximately 12 nmol/g wet weight) during the first hour. These responses correlated with differences in the ability of the two diets to increase the circulating [insulin]/[glucagon] ratio and thus to elevate the ratio of 6-phosphofructo-2-kinase to fructose-2, 6-bisphosphatase. Liver glycogen was deposited briskly in both groups of rats. To assess its mechanism of synthesis (directly from glucose versus indirectly via the gluconeogenic pathway), animals eating the chow or sucrose diets received intravenous infusions of [14C]bicarbonate, [1-14C] fructose, and 3H2O. After isolation, the glycogen was subjected to positional isotopic analysis of its glucose residues. The results established that regardless of the diet the bulk of liver glycogen was gluconeogenic in origin. The fact that with sucrose feeding carbon flow through hepatic fructose-1,6-bisphosphatase remained active despite high levels of Fru-2,6-P2 (a potent inhibitor of this enzyme in vitro) presents a metabolic paradox. Conceivably, the suppressive effect of Fru-2, 6-P2 on hepatic fructose-1,6-bisphosphatase is overridden in vivo by some unknown factor or factors generated in response to sucrose feeding. Alternatively, metabolic zonation in liver might result in the coexistence of hepatocytes rich in Fru-2,6-P2 (high glycolytic, low gluconeogenic, low glycogenic capacitites) with cells depleted of Fru-2,6-P2 (low glycolytic, high gluconeogenic, high glycogenic capacities).     

Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase.

We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a Mr of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced Mr of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,2-kinase were KmFru 6-P = 85 microM and KmATP = 270 microM, and those of fructose 2,6-bisphosphatase were KmFru 2,6-P2 = 21 microM and KiFru 6-P = 3.4 microM. The enzyme was phosphorylated by Fru-2,6[2-32P]P2 and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.     

Transgenic Arabidopsis plants with decreased activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase have altered carbon partitioning

The role of fructose-2,6-bisphosphate (Fru-2,6-P(2)) as a regulatory metabolite in photosynthetic carbohydrate metabolism was studied in transgenic Arabidopsis plants with reduced activity of Fru-6-phosphate,2-kinase/Fru-2,6-bisphosphatase. A positive correlation was observed between the Fru-6-phosphate,2-kinase activity and the level of Fru-2,6-P(2) in the leaves. The partitioning of carbon was studied by (14)CO(2) labeling of photosynthetic products. Plant lines with Fru-2,6-P(2) levels down to 5% of the levels observed in wild-type (WT) plants had significantly altered partitioning of carbon between sucrose (Suc) versus starch. The ratio of (14)C incorporated into Suc and starch increased 2- to 3-fold in the plants with low levels of Fru-2,6-P(2) compared with WT. Transgenic plant lines with intermediate levels of Fru-2,6-P(2) compared with WT had a Suc-to-starch labeling ratio similar to the WT. Levels of sugars, starch, and phosphorylated intermediates in leaves were followed during the diurnal cycle. Plants with low levels of Fru-2,6-P(2) in leaves had high levels of Suc, glucose, and Fru and low levels of triose phosphates and glucose-1-P during the light period compared with WT. During the dark period these differences were eliminated. Our data provide direct evidence that Fru-2,6-P(2) affects photosynthetic carbon partitioning in Arabidopsis. Opposed to this, Fru-2,6-P(2) does not contribute significantly to regulation of metabolite levels in darkness.     

Reciprocal changes in fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase activity in response to glucagon and epinephrine

The effect of hormones on the enzymes responsible for the synthesis (fructose-6-P,2-kinase) and degradation (fructose-2,6-Pase) of fructose-2,6-P₂ was examined in isolated rat hepatocytes. Glucagon (10^?11 M), epinephrine (10^?5 M), or calcium (2.4 mM) and A23187 (10^?5 M) administration to hepatocytes produced simultaneous activation of fructose-2,6-Pase and inactivation of fructose-6-P,2-kinase within 2 minutes. The effect of epinephrine on these two enzymes was dependent on the presence of Ca⁺⁺. These results suggest that the level of fructose-2,6-P₂ is controlled by recriprocal changes in fructose-2,6-Pase and fructose-6-P,2-kinase activities.     

Effect of modification of lysine residues of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase with pyridoxal 5'-phosphate

Inactivation of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase by pyridoxal 5'-P followed by reduction with NaBH4 was studied. Fructose-6-P,2-kinase is over 80% inactivated by 2 mM pyridoxal 5'-P. The stoichiometry of the pyridoxyl-P incorporation and the inactivation of the kinase follows a biphasic curve. The first P-pyridoxyl residue incorporated per protomer does not affect fructose-6-P,2-kinase, but the next two P-pyridoxyl incorporation/protomer results in 80% inactivation. The Km values for ATP and fructose-6-P of the enzymes containing varying amounts of P-pyridoxyl groups at intermediate levels of inactivation are not altered, but Vmax is decreased. Among the metabolites tested, only fructose-2,6-P2 and Mg-ATP are competitive with pyridoxal-P and protect the enzyme against the inactivation. Neither the activity nor the fructose-6-P inhibition of fructose-2,6-bisphosphatase is affected by the modification. The acid hydrolysate of the inactive P-[3H]pyridoxyl enzyme contained only [3H]pyridoxyl lysine. High performance liquid chromatography of tryptic peptides of phospho[3H]pyridoxyl enzymes reveals two peptides which were missing in the enzyme protected by fructose-2,6-P2 or ATP during the modification reaction. These peptides have been isolated, and their amino acid sequences have been determined as Asp-Gln-Asp-Lys-Tyr-Arg and Asp-Val-His-Lys-Tyr. Pyridoxal-P reacts specifically with two lysine residues at the fructose-2,6-P2-binding site of fructose-6-P,2-kinase but not that of fructose-2,6-bisphosphatase. The site may also overlap with the ATP-binding site.     

Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Interaction with N-bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate

N-Bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate have been tested in order to study the hexose phosphate binding sites of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase. N-Bromoacetylethanolamine phosphate is a competitive inhibitor with respect to fructose-6-P (Ki = 0.24 mM) and a noncompetitive inhibitor with ATP (Ki = 0.8 mM). The reagent inactivates fructose-6-P,2-kinase but not fructose-2,6-bisphosphatase, and the inactivation is prevented by fructose-6-P. The inactivation reaction follows pseudo first-order kinetics to completion and with increasing concentrations of N-bromoacetylethanolamine phosphate a rate saturation effect is observed. The concentration of the reagent giving the half-maximum inactivation is 2.2 mM and the apparent first order rate constant is 0.0046 s-1. The enzyme alkylated by N-bromoacetylethanolamine-P has lost over 90% of the kinase activity, retains nearly full activity of fructose-2,6-bisphosphatase, and its inhibition by fructose-6-P is not altered. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is also a competitive inhibitor of fructose-6-P,2-kinase with respect to fructose-6-P in the forward reaction and fructose-2,6-P2 in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activates fructose-2,6-bisphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate alters the fructose-2,6-P2 saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K0.5 of 0.8 microM. The reagent, however, has no effect on the fructose-6-P inhibition of the phosphatase. These results strongly suggest that hexose phosphate binding sites of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase are distinct and located in different regions of this bifunctional enzyme.     

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.     

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     

Crystal structure of the hypoxia-inducible form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3): a possible new target for cancer therapy

The hypoxia-inducible form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) plays a crucial role in the progression of cancerous cells by enabling their glycolytic pathways even under severe hypoxic conditions. To understand its structural architecture and to provide a molecular scaffold for the design of new cancer therapeutics, the crystal structure of the human form was determined. The structure at 2.1 A resolution shows that the overall folding and functional dimerization are very similar to those of the liver (PFKFB1) and testis (PFKFB4) forms, as expected from sequence homology. However, in this structure, the N-terminal regulatory domain is revealed for the first time among the PFKFB isoforms. With a beta-hairpin structure, the N terminus interacts with the 2-Pase domain to secure binding of fructose-6-phosphate to the active pocket, slowing down the release of fructose-6-phosphate from the phosphoenzyme intermediate product complex. The C-terminal regulatory domain is mostly disordered, leaving the active pocket of the fructose-2,6-bisphosphatase domain wide open. The active pocket of the 6-phosphofructo-2-kinase domain has a more rigid conformation, allowing independent bindings of substrates, fructose-6-phosphate and ATP, with higher affinities than other isoforms. Intriguingly, the structure shows an EDTA molecule bound to the fructose-6-phosphate site of the 6-phosphofructo-2-kinase active pocket despite its unfavorable liganding concentration, suggesting a high affinity. EDTA is not removable from the site with fructose-6-P alone but is with both ATP and fructose-6-P or with fructose-2,6-bisphosphate. This finding suggests that a molecule in which EDTA is covalently linked to ADP is a good starting molecule for the development of new cancer-therapeutic molecules.     

Purification and characterization of myocardial fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase

Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase and the phosphatase of the pure enzyme were 60 and 30 milliunits/mg, respectively. The molecular weight of the enzyme was 118,000, consisting of two subunits of 58,000. In some preparations of the enzyme a minor protein with a subunit Mr of 54,000 was present. This minor protein (54,000) was also bifunctional and showed the same immunoreactivity as the major protein. The specific activity of fructose-6-P,2-kinase of the minor component was three times higher than that of the major enzyme (58,000), but fructose-2,6-bisphosphatase activity was the same. These two forms have been separated by phosphocellulose chromatography. The tryptic peptide maps of these enzymes were very similar. The 58,000 enzyme was phosphorylated by cAMP-dependent protein kinase but the 54,000 enzyme was not. These results indicated that the minor 54,000 protein might be a proteolytically digested form of the 58,000 enzyme. The Km of the kinase for fructose-6-P and ATP was 70 microM and 260 microM, respectively for both the 58,000 and the 54,000 enzymes. Km for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 microM, respectively. The enzyme was phosphorylated by fructose-2,6-P2 but the stoichiometry of the phosphate incorporation was 0.05 mol/mol subunit, while 0.4 mol/mol was incorporated in rat liver enzyme under the same conditions.     

Enzyme regulation in c(4) photosynthesis : identification and localization of activities catalyzing the synthesis and hydrolysis of fructose-2,6-bisphosphate in corn leaves

Activities catalyzing the synthesis of fructose-2,6-bisphosphate (fructose-6-phosphate,2-kinase or Fru-6-P,2K) and its breakdown (fructose-2,6-bisphosphatase or Fru-2,6-P₂ase) were identified in leaves of corn (Zea mays), a C₄ plant. Fru-6-P,2K and Fru-2,6-P₂ase were both localized mainly, if not entirely, in the leaf mesophyll cells. A partially purified preparation containing the two activities revealed that the kinase and phosphatase were regulated by metabolite effectors in a manner generally similar to their counterparts in C₃ species. Thus, corn Fru-6-P,2K was activated by inorganic phosphate (Pi) and fructose-6-phosphate, and was inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fru-2,6-P₂ase was inhibited by its products, fructose-6-phosphate and Pi. However, unlike its spinach equivalent, corn Fru-2,6-P₂ase was also inhibited by 3-phosphoglycerate and, less effectively, by dihydroxyacetone phosphate. The C₄ Fru-6-P,2K and Fru-2,6-P₂ase were also quite sensitive to inhibition by phosphoenolpyruvate, and each enzyme was also selectively inhibited by certain other metabolites.     

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.