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.     

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.     

Evidence for new phosphorylation sites for protein kinase C and cyclic AMP-dependent protein kinase in bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was phosphorylated by incubation with [gamma-32P]MgATP and cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). After digestion with chymotrypsin, the phosphorylation sites for the two protein kinases were identified by peptide mapping, and microsequencing. Evidence for new phosphorylation sites for PKA (Ser-483) and PKC (Ser-84 and Ser-466) was obtained.     

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.     

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.     

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.     

A switch in the kinase domain of rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase plays an essential role in the regulation of glucose metabolism by both producing and degrading Fru-2,6-P(2) via its distinct catalytic activities. The 6-PF-2-K and Fru-2,6-P(2)ase active sites are located in separate domains of the enzyme. The kinase domain is structurally related to the superfamily of mononucleotide binding proteins that includes adenylate kinase and the G-proteins. We have determined three new structures of the enzymatic monomer, each with a different ligand in the ATP binding site of the 6-PF-2-K domain (AMP-PNP, PO(4), and water). A comparison of these three new structures with the ATPgammaS-bound 6-PF-2-K domain reveals a rearrangement of a helix that is dependent on the ligand bound in the ATP binding site of the enzyme. This helix motion dramatically alters the position of a catalytic residue (Lys172). This catalytic cation is analogous to the Arg residue donated by the rasGAP protein, and the Arg residue at the core of the GTP or GDP sensing switch motion seen in the heterotrimeric G-proteins. In addition, a succinate molecule is observed in the Fru-6-P binding site. Kinetic analysis of succinate inhibition of the 6-PF-2-K reaction is consistent with the structural findings, and suggests a mechanism for feedback inhibition of glycolysis by citric acid cycle intermediates. Alterations in the 6-PF-2-K kinetics of several proteins mutated near both the switch and the succinate binding site suggest a mode of communication between the ATP- and F6P binding sites. Together with these kinetic data, these new structures provide insights into the mechanism of the 6-PF-2-K activity of this important bifunctional enzyme.     

Cloning and expression of novel isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine heart

Distinct 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) cDNAs were cloned from bovine heart, showing that PFK-2/FBPase-2 gene B, which contains 16 exons, codes for at least five mRNAs. Three of them (B1, B2, B4) could encode the 58,000-M_r isozyme. In B2 mRNA, exon 15 encodes four more residues than in Bl. In B4 mRNA, exon 15 encodes six more residues than in B1, butexon 16 (20 residues) is missing. B3 mRNA corresponds to the 54,000-M_r isozyme. It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region. B5 mRNA encodes a truncated form. When expressed in E. coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK-2 activity.     

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.     

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.     

The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The sugar phosphate specificity of the active site of 6-phosphofructo-2-kinase and of the inhibitory site of fructose-2,6-bisphosphatase was investigated. The Michaelis constants and relative Vmax values of the sugar phosphates for the 6-phosphofructo-2-kinase were: D-fructose 6-phosphate, Km = 0.035 mM, Vmax = 1; L-sorbose 6-phosphate, Km = 0.175 mM, Vmax = 1.1; D-tagatose 6-phosphate, Km = 15 mM, Vmax = 0.15; and D-psicose 6-phosphate, Km = 7.4 mM, Vmax = 0.42. The enzyme did not catalyze the phosphorylation of 1-O-methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, 2,5-anhydro-D-mannitol 6-phosphate, D-ribose 5-phosphate, or D-arabinose 5-phosphate. These results indicate that the hydroxyl group at C-3 of the tetrahydrofuran ring must be cis to the beta-anomeric hydroxyl group and that the hydroxyl group at C-4 must be trans. The presence of a hydroxymethyl group at C-2 is required; however, the orientation of the phosphonoxymethyl group at C-5 has little effect on activity. Of all the sugar monophosphates tested, only 2,5-anhydro-D-mannitol 6-phosphate was an effective inhibitor of the kinase with a Ki = 95 microM. The sugar phosphate specificity for the inhibition of the fructose-2,6-bisphosphatase was similar to the substrate specificity for the kinase. The apparent I0.5 values for inhibition were: D-fructose 6-phosphate, 0.01 mM; L-sorbose 6-phosphate, 0.05 mM; D-psicose 6-phosphate, 1 mM; D-tagatose 6-phosphate, greater than 2 mM; 2,5-anhydro-D-mannitol 6-phosphate, 0.5 mM. 1-O-Methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, and D-arabinose 5-phosphate did not inhibit. Treatment of the enzyme with iodoacetamide decreased sugar phosphate affinity in the kinase reaction but had no effect on the sensitivity of fructose-2,6-bisphosphatase to sugar phosphate inhibition. The results suggest a high degree of homology between two separate sugar phosphate binding sites for the bifunctional enzyme.     

The kinase activity of human brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is regulated via inhibition by phosphoenolpyruvate

The two enzymatic activities of the highly conserved catalytic core of 6PF2K/Fru-2,6-P₂ase are thought to be reciprocally regulated by the amino- and carboxy-terminal regions unique to each isoform. In this study, we describe the recombinant expression, purification, and kinetic characterization of two human brain 6PF2K/Fru-2,6-P₂ase splice variants, HBP1 and HBP2. Interestingly, both lack an arginine which is highly conserved among other tissue isoforms, and which is understood to be critical to the fructose-2,6-bisphosphatase mechanism. As a result, the phosphatase activity of both HBP isoforms is negligible, but we found that it could be recovered by restoration of the arginine by site directed mutagenesis. We also found that AMP activated protein kinase and protein kinases A, B, and C catalyzed the phosphorylation of Ser-460 of HBP1, and that in addition both isoforms are phosphorylated at a second, as yet undetermined site by protein kinase C. However, none of the phosphorylations had any effect on the intrinsic kinetic characteristics of either enzymatic activity, and neither did point mutation (mimicking phosphorylation), deletion, and alternative-splice modification of the HBP1 carboxy-terminal region. Instead, these phosphorylations and mutations decreased the sensitivity of the 6PF2K to a potent allosteric inhibitor, phosphoenolpyruvate, which appears to be the major regulatory mechanism.     

Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by phenylglyoxal. Evidence for essential arginine residues.

Treatment of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with the arginine-specific reagent, phenylglyoxal, irreversibly inactivated both 6-phosphofructo-2-kinase and fructose-6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against fructose-2,6-bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 mol/mol enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic-AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme.     

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.     

Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site

Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two minor peptides were isolated from bovine heart enzyme whose amino acid sequences were Lys-Gln-Cys-Ala-Leu-Val-Ala-Leu-Lys, Arg-Ile-Glu-Cys-Tyr-Lys, and Ile-Glu-Cys-Tyr-Lys, respectively. In all cases, N-bromoacetylethanolamine-P had alkylated the cysteine residues. The amount of bromo[14C]acetylethanolamine-P incorporated into rat liver and beef heart was 1.3 mol/mol of subunit and 2.1 mol/mol of subunit, respectively, and the incorporations in the presence of Fru-6-P were reduced to 0.34 mol/mol of subunit and 0.9 mol/mol of subunit, respectively. Thus, the main fructose-6-P binding site of rat liver and bovine heart enzymes was identical except for a single amino acid substitution of valine for alanine in the latter enzyme. This peptide corresponded to residues 105 to 113 from the N terminus of the known amino acid sequence of rat liver enzyme, but since the complete sequence of bovine heart enzyme is not known, the location of the same peptide in the heart enzyme cannot be assigned.