A rat gene encoding heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

There are at least 3 isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, a bifunctional enzyme which catalyzes the synthesis and degradation of fructose 2,6-bisphosphate. A 22-kb rat gene that encodes the heart isozyme has been identified and compared with the 55-kb rat gene encoding the liver and muscle isozymes which had been described earlier. Although these 2 genes include 12 successive similar exons, they contain dissimilar exons at both ends, consistent with the occurrence of different regulatory domains at the N- and C-termini in the 3 isozymes.     

Characterization of an enhancer upstream from the muscle-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The muscle-type isozyme of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is encoded by a mRNA transcribed from the M promoter of a 55-kb gene, which also produces the liver-type isozyme from an alternative promoter. By transient transfection and in vitro protein-DNA binding assays we have delineated, within 4.7 kb of 5' flanking sequence, the M promoter proper and an enhancer located between -1615 and -1809. This enhancer stimulated up to 12-fold the activity of the promoter in the context of an intact 5' flanking sequence and close to 900-fold the activity of the minimal (+41 to -40) M promoter cloned directly downstream from it. A functional dissection of the enhancer by site-directed mutagenesis and use of oligonucleotides suggested that its activity involves the cooperative effect of six binding sites for trans-acting factors clustered within 150 bp. These sites contain either an EF-1A/E4TF1 motif (also known to bind the ets oncogene product) or a Sp1 motif, or both. The activity of the enhancer could be demonstrated in L6 myoblasts and myocytes and in FTO2B hepatoma cells. When left within the intact 5' flanking sequence, however, enhancer activity was inhibited upon differentiation of myoblasts into myocytes.     

Binding of ATP to the fructose-2,6-bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase leads to activation of its 6-phosphofructo-2-kinase

To understand the mechanism by which the activity of the 6-phosphofructo-2-kinase (6PF-2K) of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is stimulated by its substrate ATP, we studied two mutants of the enzyme. Mutation of either Arg-279, the penultimate basic residue within the Walker A nucleotide-binding fold in the bisphosphatase domain, or Arg-359 to Ala eliminated the activation of the chicken 6PF-2K by ATP. Binding analysis by fluorescence spectroscopy using 2'(3')-O-(N-methylanthraniloyl)-ATP revealed that the kinase domains of these two mutants, unlike that of the wild type enzyme, showed no cooperativity in ATP binding and that the mutant enzymes possess only the high affinity ATP binding site, suggesting that the ATP binding site on the bisphosphatase domain represents the low affinity site. This conclusion was supported by the result that the affinity of ATP for the isolated bisphosphatase domain is similar to that for the low affinity site in the wild type enzyme. In addition, we found that the 6PF-2K of a chimeric enzyme, in which the last 25 residues of chicken enzyme were replaced with those of the rat enzyme, could not be activated by ATP, despite the fact that the ATP-binding properties of this chimeric enzyme were not different from those of the wild type chicken enzyme. These results demonstrate that activation of the chicken 6PF-2K by ATP may result from allosteric binding of ATP to the bisphosphatase domain where residues Arg-279 and Arg-359 are critically involved and require specific C-terminal sequences.     

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.     

Hormonal regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: kinetic models

A graphical method to reveal the so-called 'critical fragments' in schemes of biochemical systems is considered. These fragments produce multiple steady states or self-oscillations in systems. As an example, the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, regulated by glucagon through enzyme phosphorylation, is discussed. It is shown that this enzyme may act as a metabolic switching mechanism in discontinuous or oscillatory regimes, depending on the specific structure of its kinetic scheme. The boundaries of concentrational and parameter domains for these critical phenomena are also predicted.     

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.     

Arg-257 and Arg-307 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6-bisphosphate in the fructose-2,6-bisphosphatase domain.

Rat liver fructose-2,6-bisphosphatase, which catalyzes its reaction via a phosphoenzyme intermediate, is evolutionarily related to the phosphoglycerate mutase enzyme family (Bazan, F., Fletterick, R., and Pilkis, S.J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). Arg-7 and Arg-59 of the yeast phosphoglycerate mutase have been postulated to be substrate-binding residues based on the x-ray crystal structure. The corresponding residues in rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, Arg-257 and Arg-307, were mutated to alanine. The Arg257Ala and Arg307Ala mutants and the wild-type enzyme were expressed in Escherichia coli and then purified to homogeneity. Both mutant enzymes had identical far and near UV circular dichroism spectra and 6-phosphofructo-2-kinase activities when compared with the wild-type enzyme. However, the Arg257Ala and Arg307Ala mutants had altered steady state fructose-2,6-bisphosphatase kinetic properties; the Km values for fructose-2,6-bisphosphate of the Arg257Ala and Arg307Ala mutants were increased by 12,500- and 760-fold, whereas the Ki values for inorganic phosphate were increased 7.4- and 147-fold, respectively, as compared with the wild-type values. However, the Ki values for the other product, fructose-6-phosphate, were unchanged for the mutant enzymes. Although both mutants exhibited parallel changes in kinetic parameters that reflect substrate/product binding, they had opposing effects on their respective maximal velocities; the maximal velocity of Arg257Ala was 11-fold higher, whereas that for Arg307Ala was 700-fold lower, than that of the wild-type enzyme. Pre-steady state kinetic studies demonstrated that the rate of phosphoenzyme formation for Arg307Ala was at least 4000-fold lower than that of the wild-type enzyme, whereas the rate for Arg257Ala was similar to the wild-type enzyme. Furthermore, consistent with the Vmax changes, the rate constant for phosphoenzyme breakdown for Arg257Ala was increased 9-fold, whereas that for Arg307Ala was decreased by a factor of 500-fold, as compared with the wild-type value. The results indicate that both Arg-257 and Arg-307 interact with the reactive C-2 phospho group of fructose 2,6-bisphosphate and that Arg-307 stabilizes this phospho group in the transition state during phosphoenzyme breakdown, whereas Arg-257 stabilizes the phospho group of the ground state phosphoenzyme intermediate.     

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.     

Cloning, expression and chromosomal localization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene

6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional enzyme responsible for the synthesis and breakdown of Fru-2,6-P2, a key metabolite in the regulation of glycolysis. Several genes encode distinct PFK-2/FBPase-2 isozymes that differ in their tissue distribution and enzyme regulation. In this paper, we present the isolation of a cDNA from a human testis cDNA library that encodes a PFK-2/FBPase-2 isozyme. Sequencing data show an open reading frame of 1407 nucleotides that codifies for a protein of 469 amino acids. This has a calculated molecular weight of 54kDa and 97% similarity with rat testis PFK-2/FBPase-2, with complete conservation of the amino acid residues involved in the catalytic mechanism. Fluorescence in-situ hybridization (FISH) localized testis PFK-2/FBPase-2 gene (PFKFB4) in human chromosome 3 at bands p21-p22. A Northern blot analysis of different rat tissues showed the presence of a 2.4-kb mRNA expressed specifically in testis. In mammalian COS-1 cells, the human testis cDNA drives expression of an isozyme with a molecular weight of 55kDa. This isozyme shows clear PFK-2 activity. Taken together, these results provide evidence for a new PFK-2/FBPase-2 gene coding for a human testis isozyme.     

Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves.

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase specific activity 10.4-15.9 nmol min(-1) x mg protein (-1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmol x mol(-1) mg protein(-1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.     

Complete amino acid sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The complete amino acid sequence of 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was determined by direct analysis of the S-carboxamidomethyl protein. A complete set of nonoverlapping peptides was produced by cleavage with a combination of cyanogen bromide and specific proteolytic enzymes. The active enzyme is a dimer of two identical polypeptide chains composed of 470 amino acids each. The NH2-terminal amino acid residue of the polypeptide chain was shown to be N-acetylserine by fast atom bombardment mass spectrometry of the purified N-terminal tetradecapeptide isolated after cleavage of the intact S-carboxamidomethylated protein with lysyl endoproteinase (Achromobacter protease I). Alignment of the set of unique peptides was accomplished by the analysis of selected overlapping peptides generated by proteolytic cleavage of the intact protein and the larger purified cyanogen bromide peptides with trypsin, Staphylococcus aureus V8 protease, and lysyl endoproteinase. Four nonoverlapping peptides were aligned by comparison with the amino acid sequence predicted from a partial cDNA clone encoding amino acid positions 166-470 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Colosia, A.D., Lively, M., El-Maghrabi, M. R., and Pilkis, S. J. (1987) Biochem. Biophys. Res. Commun. 143, 1092-1098). The nucleotide sequence of the cDNA corroborated the peptide sequence determined by direct methods. A search of the Protein Identification Resource protein sequence database revealed that the overall amino acid sequence appears to be unique since no obviously homologous sequences were identified. However, a 100-residue segment of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (residues 250-349), including the active site histidine residue of the bisphosphatase domain, was found to be homologous to the active site regions of yeast phosphoglycerate mutase and human bisphosphoglycerate mutase.