Bovine brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Evidence for a neural-specific isozyme.

Bovine brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was purified to homogeneity and characterized. This bifunctional enzyme is a homodimer with a subunit molecular weight of 120,000, which is twice that of all other known bifunctional enzyme isozymes. The kinase/bisphosphatase activity ratio was 3.0. The Km values for fructose 6-phosphate and ATP of the 6-phosphofructo-2-kinase were 27 and 55 microM, respectively. The Km for fructose 2,6-bisphosphate and the Ki for fructose 6-phosphate for the bisphosphatase were 70 and 20 microM, respectively. Physiologic concentrations of citrate had reciprocal effects on the enzyme's activities, i.e. inhibiting the kinase (Ki of 35 microM) and activating the bisphosphatase (Ka of 16 microM). Phosphorylation of the brain enzyme was catalyzed by the cyclic AMP-dependent protein kinase with a stoichiometry of 0.9 mol of phosphate/mol of subunit and at a rate similar to that seen with the liver isozyme. In contrast to the liver isozyme, the kinetic properties of the brain enzyme were unaffected by cyclic AMP-dependent protein kinase phosphorylation, and also was not a substrate for protein kinase C. The brain isozyme formed a labeled phosphoenzyme intermediate and cross-reacted with antibodies raised against the liver isozyme. However, the NH2-terminal amino acid sequence of a peptide generated by cyanogen bromide cleavage of the enzyme had no identity with any known bifunctional enzyme sequences. These results indicate that a novel isozyme, which is related to other 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes, is expressed specifically in neural tissues.     

Functional homology of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, phosphoglycerate mutase, and 2,3-bisphosphoglycerate mutase

The bisphosphatase domain of the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to exhibit a structural similarity to yeast phosphoglycerate mutase and human red blood cell 2,3-bisphosphoglycerate mutase including very similar active site sequences with a histidyl residue being involved in phospho group transfer. The liver bifunctional enzyme was found to catalyze the hydrolysis of glycerate 1,3-bisphosphate to glycerate 3-phosphate and inorganic phosphate. The Km for glycerate 1,3-bisphosphate was 320 microM and the Vmax was 11.5 milliunits/mg. Incubation of the rat liver enzyme with [1-32P]glycerate 1,3-bisphosphate resulted in the formation of a phosphoenzyme intermediate, and the labeled amino acid was identified as 3-phosphohistidine. Tryptic and endoproteinase Lys-C peptide maps of the 32P-phosphoenzyme labeled either with [2-32P]fructose 2,6-bisphosphate or [1-32P]glycerate 1,3-bisphosphate revealed that 32P-radioactivity was found in the same peptide, proving that the same histidyl group accepts phosphate from both substrates. Fructose 2,6-bisphosphate inhibited competitively the formation of phosphoenzyme from [1-32P]glycerate 1,3-bisphosphate. Effectors of fructose-2,6-bisphosphatase also inhibited phosphoenzyme formation. Substrates and products of phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase also modulated the activities of the bifunctional enzyme. These results demonstrate that, in addition to a structural homology, the bisphosphatase domain of the bifunctional enzyme has a functional similarity to phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase and support the concept of an evolutionary relationship between the three enzyme activities.     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by o-phthalaldehyde.

The two activities of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by o-phthalaldehyde. Absorbance and fluorescence spectra of the modified enzyme were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme subunit). The inactivation of 6-phosphofructo-2-kinase by o-phthalaldehyde was faster than the inactivation of fructose-2,6-bisphosphatase, which was concomitant with the increase in fluorescence. The substrates of 6-phosphofructo-2-kinase did not protect the kinase against inactivation, whereas fructose-2,6-bisphosphate fully protected against o-phthalaldehyde-induced inactivation of the bisphosphatase. Addition of dithiothreitol prevented both the increase in fluorescence and the inactivation of fructose-2,6-bisphosphatase, but not that of 6-phosphofructo-2-kinase. It is proposed that o-phthalaldehyde forms two different inhibitory adducts: a non-fluorescent adduct in the kinase domain and a fluorescent isoindole derivative in the bisphosphatase domain. A lysine and a cysteine residue could be involved in fructose-2,6-bisphosphate binding in the bisphosphatase domain of the protein.     

Effects of various metabolic conditions and of the trivalent arsenical melarsen oxide on the intracellular levels of fructose 2,6-bisphosphate and of glycolytic intermediates in Trypanosoma brucei

Upon differential centrifugation of cell-free extracts of Trypanosoma brucei, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase behaved as cytosolic enzymes. The two activities could be separated from each other by chromatography on both blue Sepharose and anion exchangers. 6-phosphofructo-2-kinase had a Km for both its substrates in the millimolar range. Its activity was dependent on the presence of inorganic phosphate and was inhibited by phosphoenolpyruvate but not by citrate or glycerol 3-phosphate. The Km of fructose-2,6-bisphosphatase was 7 microM; this enzyme was inhibited by fructose 1,6-bisphosphate (Ki = 10 microM) and, less potently, by fructose 6-phosphate, phosphoenolpyruvate and glycerol 3-phosphate. Melarsen oxide inhibited 6-phosphofructo-2-kinase (Ki less than 1 microM) and fructose-2,6-bisphosphatase (Ki = 2 microM) much more potently than pyruvate kinase (Ki greater than 100 microM). The intracellular concentrations of fructose 2,6-bisphosphate and hexose 6-phosphate were highest with glucose, intermediate with fructose and lowest with glycerol and dihydroxyacetone as glycolytic substrates. When added with glucose, salicylhydroxamic acid caused a decrease in the concentration of fructose 2,6-bisphosphate, ATP, hexose 6-phosphate and fructose 1,6-bisphosphate. These studies indicate that the concentration of fructose 2,6-bisphosphate is mainly controlled by the concentration of the substrates of 6-phosphofructo-2-kinase. The changes in the concentration of phosphoenolpyruvate were in agreement with the stimulatory effect of fructose 2,6-bisphosphate on pyruvate kinase. At micromolar concentrations, melarsen oxide blocked almost completely the formation of fructose 2,6-bisphosphate induced by glucose, without changing the intracellular concentrations of ATP and of hexose 6-phosphates. At higher concentrations (3-10 microM), this drug caused cell lysis, a proportional decrease in the glycolytic flux, as well as an increase in the phosphoenolypyruvate concentrations which was restricted to the extracellular compartment. Similar changes were induced by digitonin. It is concluded that the lytic effect of melarsen oxide on the bloodstream form of T. brucei is not the result of an inhibition of pyruvate kinase.     

Purification and characterization of fructose-2,6-bisphosphatase, a substrate-specific cytosolic enzyme from leaves

Fructose-2,6-bisphosphatase (EC 3.1.3.46), which hydrolyzes fructose 2,6-bisphosphate to fructose 6-phosphate and Pi, has been purified to apparent homogeneity from spinach leaves and found to be devoid of fructose-6-phosphate,2-kinase activity. The isolated enzyme is a dimer (76 kDa determined by gel filtration) composed of two 33-kDa subunits. The enzyme is highly specific and displays hyperbolic kinetics with its fructose 2,6-bisphosphate substrate (Km = 32 microM). The products of the reaction, fructose 6-phosphate and Pi, along with AMP and Mg2+ are inhibitors of the enzyme. Nonaqueous cell fractionation revealed that, like the fructose 2,6-bisphosphate substrate, fructose-2,6-bisphosphatase as well as fructose-6-phosphate,2-kinase occur in the cytosol of spinach leaves.     

The effect of arabinose 1,5-bisphosphate on rat hepatic 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase

The alpha- and beta-anomers of arabinose 1,5-bisphosphate and ribose 1,5-bisphosphate were tested as effectors of rat liver 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase. Both anomers of arabinose 1,5-bisphosphate activated the kinase and inhibited the bisphosphatase. The alpha-anomer was the more effective kinase activator while the beta-anomer was the more potent inhibitor of the bisphosphatase. Inhibition of the bisphosphatase by both anomers was competitive, and both potentiated allosteric inhibition by AMP. beta-Arabinose 1,5-bisphosphate was also more effective in decreasing fructose 2,6-bisphosphate binding to the enzyme. Neither anomer of ribose 1,5-bisphosphate affected 6-phosphofructo-1-kinase or fructose-1,6-bisphosphatase, indicating that the configuration of the C-2 (C-3 in Fru 2,6-P2) hydroxyl group is important for biological activity. These results are also consistent with arabinose 1,5-bisphosphate binding to the active site and thereby enhancing the interaction of AMP with the allosteric site.     

Occurrence and characterization of fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase in Euglena gracilis

1. Fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase occurred in Euglena gracilis SM-ZK, and is located in cytosol. 2. Fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase were partially purified, and both enzyme activities were not separated during the partial purification. 3. The pH optimum for fructose 6-phosphate, 2-kinase activity was 7.0. The saturation curve of the enzyme activity for ATP concentration was hyperbolic, and the Km value for the substrate was 0.88 mM. On the other hand, the saturation curve of the enzyme activity for fructose 6-phosphate concentration was sigmoidal, and the K0.5 value for the substrate was 70 microM. 4. The pH optimum for fructose 2,6-bisphosphatase activity was 6.5. The saturation curve for fructose 2,6-bisphosphate concentration was sigmoidal, and the K0.5 value for the substrate was 1.29 microM. Fructose 2,6-bisphosphate showed a substrate inhibition at high concentration over 5 microM, and the enzyme activity was completely inhibited by 20 microM of fructose 2,6-bisphosphate.     

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.     

Regulation of rat liver fructose 2,6-bisphosphatase

An enzyme activity that catalyzes the hydrolysis of phosphate from the C-2 position of fructose 2,6-bisphosphate has been detected in rat liver cytoplasm. The S0.5 for fructose 2,6-bisphosphate was about 15 microM and the enzyme was inhibited by fructose 6-phosphate (Ki 40 microM) and activated by Pi (KA 1 mM). Fructose 2,6-bisphosphatase activity was purified to homogeneity by specific elution from phosphocellulose with fructose by specific elution from phosphocellulose with fructose 6-phosphate and had an apparent molecular weight of about 100,000, 6-phosphofructo 2-kinase activity copurified with fructose 2,6-bisphosphatase activity at each step of the purification scheme. Incubation of the purified protein with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in the incorporation of 1 mol of 32P/mol of enzyme subunit (Mr = 50,000). Concomitant with this phosphorylation was an activation of the fructose 2,6-bisphosphatase and an inhibition of the 6-phosphofructo 2-kinase activity. Glucagon addition to isolated hepatocytes also resulted in an inhibition of 6-phosphofructo 2-kinase and activation of fructose 2,6-bisphosphatase measured in cell extracts, suggesting that the hormone regulates the level of fructose 2,6-bisphosphate by affecting both synthesis and degradation of the compound. These findings suggest that this enzyme has both phosphohydrolase and phosphotransferase activities i.e. that it is bifunctional, and that both activities can be regulated by cAMP-dependent phosphorylation.     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The role of surface loop basic residues in substrate binding to the fructose-2,6-bisphosphatase domain.

Lys-356 has been implicated as 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 (Li, L., Lin, K., Correia, J., and Pilkis, S. J. (1992) J. Biol. Chem. 267, 16669-16675). To ascertain whether the three other basic residues (Arg-352, Arg-358, and Arg-360), which are located in a surface loop (residues 331-362) which contains Lys-356, are important in substrate binding, these arginyl residues were mutated to Ala, and each arginyl mutant was expressed in Escherichia coli and purified to homogeneity. The far UV circular dichroism spectra of the mutants were identical to that of the wild-type enzyme. The kinetic parameters of 6-phosphofructo-2-kinase of the mutants revealed only small changes. However, the Km for fructose 2,6-bisphosphate, Ki for fructose 6-phosphate, and Ka for inorganic phosphate of fructose-2,6-bisphosphatase for Arg352Ala were, respectively, 2,800-, 4,500-, and 1,500-fold higher than those for the wild-type enzyme, whereas there was no change in the maximal velocity or the Ki for inorganic phosphate. The Km for fructose 2,6-bisphosphate and Ki for inorganic phosphate of Arg360Ala were 10- and 12-fold higher, respectively, than those of the wild-type enzyme, whereas the maximal velocity and Ki for fructose 6-phosphate were unchanged. In addition, substrate inhibition was not observed with Arg352Ala and greatly reduced with Arg360Ala. The properties of the Arg358Ala mutant were identical to those of the wild-type enzyme. The results demonstrate that in addition to Lys-356, Arg-352 is another critical residue in fructose-2,6-bisphosphatase for binding the C-6 phospho group of fructose 2,6-bisphosphate and that Arg-360 binds the C-2 phospho group of fructose 2,6-bisphosphate in the phosphoenzyme.fructose 2,6-bisphosphate complex. The results also provide support for Arg-352, Lys-356, and Arg-360 constituting a specificity pocket for fructose-2,6-bisphosphatase.     

Amino acid sequence of the phosphorylation site of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was phosphorylated by cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Treatment of the 32P-labeled enzyme with thermolysin removed all of the radioactivity from the enzyme core and produced a single labeled peptide. The phosphopeptide was purified by ion exchange chromatography, gel filtration, and reverse phase high pressure liquid chromatography. The sequence of the 12-amino acid peptide was found to be Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser(P)-Ser-Ile-Pro-Gln. Correlation of the extent of phosphorylation with activity showed that a 50% decrease in the ratio of kinase activity to bisphosphate activity occurred when only 0.25 mol of phosphate was incorporated per mol of enzyme subunit, and maximal changes occurred with 0.7 mol incorporated. The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of the native bifunctional enzyme was compared with that of other rat liver protein substrates. The Km for 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (10 microM) was less than that for rat liver pyruvate kinase (39 microM), fructose-1,6-bisphosphatase (222 microM), and 6- phosphofructose -1-kinase (230 microM). Comparison of the initial rate of phosphorylation of a number of protein substrates of the cyclic AMP-dependent protein kinase revealed that only skeletal muscle phosphorylase kinase was phosphorylated more rapidly than the bifunctional enzyme. Skeletal muscle glycogen synthase, heart regulatory subunit of cyclic AMP-dependent protein kinase, and liver pyruvate kinase were phosphorylated at rates nearly equal to that of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, while phosphorylation of fructose-1,6-bisphosphatase and 6-phosphofructo-1-kinase was barely detectable. Phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was not catalyzed by any other protein kinase tested. These results are consistent with a primary role of the cyclic AMP-dependent protein kinase in regulation of the enzyme in intact liver.     

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: phosphate dependence and effects of other oxyanions

The effects of various oxyanions on the activities of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) were examined. No evidence could be found for an absolute dependence of the kinase activity on inorganic phosphate as was recently reported by M. Laloux, E. Van Schaftingen, and H.-G. Hers ((1985) Eur. J. Biochem. 148, 155-159). Rather, phosphate and arsenate activated the kinase by decreasing the enzyme's Km for fructose 6-phosphate without affecting its Km for ATP or Vmax. The Km of the kinase for fructose 6-phosphate in the presence of inorganic phosphate was found to be significantly lower (6 microM) than previously reported (30 microM) when the hydrolysis of fructose 2,6-bisphosphate by the concomitant bisphosphatase activity at low Fru 6-P concentrations was taken into account. The KA's for phosphate and arsenate activation of the kinase were 0.2 and 0.3 mM, respectively. A number of other oxyanions, including pyrophosphate, sulfate, tungstate, selenate, and molybdate all inhibited the kinase by increasing the Km for fructose 6-phosphate. The apparent Ki's for inhibition of the kinase were in the 0.5-1 mM range. In contrast, all of these oxyanions activated the bisphosphatase, with half-maximal effects requiring millimolar concentrations. Inorganic phosphate was the most potent activator with a KA of 1 mM. In contrast to the other oxyanions, vanadate and meta-periodate inhibited the kinase but had no effect on the bisphosphatase. Vanadate appeared to be a noncompetitive inhibitor since its effects were not overcome by Pi, ATP, or fructose 6-phosphate, and the species responsible was shown to be decavanadate. Like vanadate, meta-periodate had no effect on the bisphosphatase, though it was a potent inhibitor (I0.5 = 30 microM) of the kinase. Its effects were shown to be time-dependent and reversed by dithiothreitol, suggesting that it acted by an oxidative mechanism. These results augment the mounting body of evidence that the enzyme's two reactions are catalyzed at discrete active sites.     

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     

Rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a review of relationships between the two activities of the enzyme

Both the synthesis and the degradation of Fru-2,6-P2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady-state concentration of fructose 2,6-P2, an activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors. Recent evidence suggests that both reactions may proceed by two-step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6-P and fructose 2,6-P2. A labeled phosphoenzyme is formed rapidly during incubation with [2-32P]Fru-2,6-P2. The labeled residue has been identified as 3-phosphohistidine. However, it was not possible to demonstrate significant labeling of the enzyme directly from [gamma-32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a fructose 2,6-bisphosphatase site which is readily phosphorylated by fructose 2,6-P2. Additional evidence in support of two active sites include: limited proteolysis with thermolysin results in loss of 6-phosphofructo 2-kinase activity and activation of fructose 2,6-bisphosphatase, mixed function oxidation results in inactivation of the 6-phosphofructo 2-kinase but no affect on the fructose 2,6-bisphosphatase, N-ethylmaleimide treatment also inactivates the kinase but does not affect the bisphosphatase, and p-chloromercuribenzoate immediately inactivates the fructose 2,6-bisphosphatase but not the 6-phosphofructo 2-kinase. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine-synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PII in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.     

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.     

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.     

Effect of sulfhydryl modification on the activities of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Alkylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with p-mercuribenzoate caused a rapid stimulation of the kinase and an inhibition of the bisphosphatase. At later times, the kinase activity also became inhibited. In contrast, treatment with N-ethylmaleimide abolished kinase activity but had no effect on the bisphosphatase. Selective modification of residues involved in the kinase reaction was also seen with iodoacetamide, which caused a 10-fold stimulation of the kinase Vmax without affecting the bisphosphatase. The stimulatory effect of carboxyamidomethylation was seen when the kinase was assayed in the presence of inorganic phosphate, an allosteric activator of the enzyme. The iodoacetamide-treated enzyme had a 10-20-fold higher Km for fructose 6-phosphate than the native enzyme and the Ki for fructose 2,6-bisphosphate was also increased. However, the adenine-nucleotide site did not seem to be affected since there was no change in the Km for ATP, the Ki for ADP, or the adenine-nucleotide exchange. There was also a direct correlation between the incorporation of [14C]acetamide into the enzyme and activation of the kinase. The residues modified by iodoacetamide were shown to be cysteines by the exclusive appearance of carboxymethylcysteine in protein hydrolysates. Activation was associated with alkylation of 2 cysteines/subunit, of the 12 which could be alkylated after denaturation/reduction. Iodoacetamide-activated kinase was inhibited by ascorbate/Fe3+, which has been shown to modify sulfhydryl groups in the native enzyme, with concomitant loss of kinase activity.     

Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site affects phosphate activation.

To identify those residues involved in fructose 6-phosphate binding to the kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase site-directed mutations were engineered at Lys194, Arg195, Arg230, and Arg238. The mutant enzymes were purified to homogeneity by anion exchange and Blue-Sepharose chromatography and/or substrate elution from phosphocellulose columns. Circular dichroism experiments demonstrated that all of the single amino acid mutations had no effect on the secondary structure of the protein. In addition, when fructose-2,6-bisphosphatase activity was measured, all mutants had Km values for fructose 2,6-bisphosphate, Ki values for fructose 6-phosphate, and maximal velocities similar to that of the wild-type enzyme. Mutation of Arg195----Ala, or His, had little or no effect on the maximal velocity of the kinase but increased the Km for fructose 6-phosphate greater than 3,000-fold. Furthermore, the Ka for phosphate for Arg195Ala was increased 100-fold compared with the wild-type enzyme. Mutation of Lys194----Ala had no effect on maximal velocity or the Km for fructose 6-phosphate. Mutation of either Arg230 or Arg238----Ala increased the maximal velocity and the Km for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the Km values for ATP of the Arg230Ala and Arg238Ala mutants were 30-40-fold higher than that for the wild-type enzyme. Mutation of Gly48----Ala resulted in a form with no kinase activity, but fructose-2,6-bisphosphatase activity was identical to that of the wild-type enzyme. The results indicate that: 1) Arg195 is a critical residue for the binding of fructose 6-phosphate to the 6-phospho-fructo-2-kinase domain, and that interaction of the sugar phosphate with Arg195 is highly specific since mutation of the adjacent Lys194----Ala had no effect on fructose 6-phosphate binding; 2) Arg195 also play an important role in the binding of inorganic phosphate; and 3) Gly48 is an important residue in the nucleotide binding fold of 6-phosphofructo-2-kinase and that both Arg230 and Arg238 are also involved in ATP binding; and 4) the bifunctional enzyme has two separate and independent fructose 6-phosphate binding sites.     

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.