Cellular Distribution of 6-Phosphofructo-1-Kinase Isoenzymes in Rat Brain

Abstract: In the brain, all three isoenzyme types [muscle (M), liver (L), and brain (C)] of 6-phosphofructo-1-kinase (PFK; EC 2.7.1.11) occur, forming a complex mixture of homo- and heterotetramers. The PFK isoenzyme pattern of the different brain cell types is yet unknown. In the present study, we investigated the distribution of the PFK isoenzyme subunits in primary and secondary cell cultures and in bulk-isolated cells of rat brain by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. All three PFK isoenzymes are present in all cell types but in different proportions. The cellular distribution of the PFK isoenzymes in situ was studied immunohistochemically with different polyclonal antisera against purified rat PFKs. The monospecific antibody against M-type PFK stained preferentially the perinuclear areas of neurons and glial cells. The antibodies that in immunoblots detected mainly the L-type PFK showed a characteristic staining in only the cytoplasma and the processes of cells, whereas the C-type antibodies almost homogeneously stained whole cell bodies as well as large dendrites. Because the PFK isoenzymes differ with respect to their allosteric properties, their differential distribution in different brain cells might be of importance for the regulation of brain glycolysis in the different cellular compartments of the brain.     

Identification of allosteric sites in rabbit phosphofructo-1-kinase

Earlier studies indicated an evolutionary relationship between bacterial and mammalian phosphofructo-1-kinases (PFKs) that suggests duplication, tandem fusion, and divergence of catalytic and effector binding sites of a prokaryotic ancestor to yield in eukaryotes a total of six organic ligand binding sites. The identities of residues involved in the four binding sites for allosteric ligands in mammalian PFK have been inferred from this assumed relationship. In the current study of the C isozyme of rabbit PFK, two arginine residues that can be aligned with important residues in the catalytic and allosteric binding sites of bacterial PFK and that are conserved in all eukaryotic PFKs were mutated. Arg-48 was suggested previously to be part of either the ATP inhibitory or the adenine nucleotide activating site. However, the mutant enzyme showed only slightly less sensitivity to ATP inhibition and was fully activatable by adenine nucleotides. On the other hand, sensitivity to citrate and 3-phosphoglycerate inhibition was lost, indicating an important role for Arg-48 in the binding of these allosteric effectors. Mutation of Arg-481, homologous to an active site residue in bacterial PFK, prevented binding and allosteric activation by fructose 2,6-bisphosphate. A new relationship between the allosteric sites of mammalian PFK and bacterial PFK is proposed.     

Inorganic pyrophosphate: fructose-6-phosphate 1-phosphotransferase of the potato tuber is related to the major ATP-dependent phosphofructokinase of E. coli

A procedure was developed for the purification of inorganic pyrophosphate: fructose-6-phosphate 1-phospho-transferase (PPi-PFK) from potato tubers. The enzyme has the structure alpha 4 beta 4 with a subunit of 68 kDa and a beta subunit of 60 kDa. The structural relationship of this enzyme to other PFKs and to fructose bisphosphatase was examined by immunoprecipitation and immunoblotting. Antibodies to the plant enzyme did not react with E. coli PFK. No cross-reaction was seen among the following enzymes or their antibodies: yeast fructose bisphosphatase; rabbit PFKs A, B, or the enzyme from brain; and the two subunits of the potato PPi-PFK. On the other hand, antibody to E. coli PFK-1 strongly cross-reacts with the 60 kDa polypeptide but not 68 kDa peptide.     

Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.

The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition.     

Liver peptide stabilizing factor protects phosphofructokinase against inactivation by fructose-1,6-bisphosphatase.

Rabbit liver fructose-1,6-bisphosphatase (FDPase) can reversibly inactivate both rabbit muscle and rat liver phosphofructokinases (PFK) under appropriate conditions. The peptide factor which stabilizes rat liver PFK-L₂ against thermal inactivation has now been found to protect both PFKs from inactivation by FDPase. Assay at high ATP (ca. 3 mM) is necessary to demonstrate these reversible changes. In addition, the activation of FDPase by liver cytosol, by oleate plus cytosol, or by oleate plus muscle PFK is lowere about 50% in the presence of peptide factor. These observations suggest an active participation of the peptide factor in regulation of liver glycolysis and gluconeogenesis.     

Leishmania donovani phosphofructokinase. Gene characterization, biochemical properties and structure-modeling studies.

The characterization of the gene encoding Leishmania donovani phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported. L. donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53 988 and a pI of 9.26. The predicted amino acid sequence contains a C-terminal tripeptide that conforms to an established signal for glycosome targeting. L. donovani PFK showed most sequence similarity to inorganic pyrophosphate (PPi)-dependent PFKs, despite being ATP-dependent. It thereby resembles PFKs from other Kinetoplastida such as Trypanosoma brucei, Trypanoplasma borreli (characterized in this study), and a PFK found in Entamoeba histolytica. It exhibited hyperbolic kinetics with respect to ATP whereas the binding of the other substrate, fructose 6-phosphate, showed slight positive cooperativity. PPi, even at high concentrations, did not have any effect. AMP acted as an activator of PFK, shifting its kinetics for fructose 6-phosphate from slightly sigmoid to hyperbolic, and increasing considerably the affinity for this substrate, whereas GDP did not have any effect. Modelling studies and site-directed mutagenesis were employed to shed light on the structural basis for the AMP effector specificity and on ATP/PPi specificity among PFKs.     

Aurintricarboxylic acid is a potent inhibitor of phosphofructokinase.

Aurintricarboxylic acid (ATA) was found to be a very potent inhibitor of purified rabbit liver phosphofructokinase (PFK), giving 50% inhibition at 0.2 microM. The inhibition was in a manner consistent with interaction at the citrate-inhibitory site of the enzyme. The data suggest that inhibition of PFK by ATA was not due to denaturation of the enzyme or the irreversible binding of inhibitor, since the inhibition could be reversed by addition of allosteric activators of PFK, i.e. fructose 2,6-bisphosphate or AMP. Two other tricarboxylic acids, agaric acid and (-)-hydroxycitrate, were found to inhibit PFK. ATA at much higher concentrations (500 microM) was shown to inhibit fatty acid synthesis from endogenous glycogen in rat hepatocytes; however, protein synthesis was not altered.     

Comparison of phosphofructokinases in submandibular glands of immature and adult rats

1. Phosphofructokinases (PFKs) in immature and adult rat submandibular glands were purified to near homogeneity, and their properties were compared. 2. PFK in immature gland was less sensitive to inhibition by ATP than adult PFK. 3. Saturation curve for fructose 6-phosphate of PFK in immature gland was less sigmoidal than that of adult PFK indicating the lower cooperativity of subunits in immature PFK. 4. Fructose 2,6-bisphosphate relieved PFK from inhibition by ATP in adult gland, but a similar effect was not clearly observed in immature gland PFK. 5. Adult PFK was a heterotetramer consisting of C-, M-, L-subunits, but in immature PFK another type of subunit, which was slightly smaller than L-subunit, existed in addition to C-, M- and L-subunits.     

Evolution of the allosteric ligand sites of mammalian phosphofructo-1-kinase

Mammalian phosphofructokinase (PFK) has evolved by a process of tandem gene duplication and fusion to yield a protein that is more than double the size of prokaryotic PFKs. On the basis of complete conservation of active site residues in the N-terminal half of the eukaryotic enzyme with those of the bacterial PFKs, one assumes that the active site of the eukaryotic PFK is located in the N-terminal half. Again using sequence comparisons, the four allosteric ligand sites of mammalian PFK have been thought to arise from the duplicated catalytic and regulatory sites of the ancestral PFK. Previous site-directed mutagenesis studies [Li et al. (1999) Biochemistry 38, 16407-16412; Chang and Kemp (2002) Biochem. Biophys. Res. Commun. 290, 670-675] have identified the origins of the citrate and fructose 2,6-bisphosphate sites. Here, site-directed mutagenesis of two arginine residues (Arg-433 and Arg-429) of mouse phosphofructokinase is used to identify the ATP inhibitory site, and, by inference, the AMP/ADP site. Mutation of the residues to alanine reduced ATP inhibition in the case of Arg-429 and eliminated ATP inhibition in the instance of Arg-433. The Arg-433 mutant could be inhibited by citrate, and that inhibition could be reversed by fructose 2,6-bisphosphate and cyclic AMP, a high-affinity ligand for the AMP/ADP binding site. It is concluded that the two inhibitors, ATP and citrate, of mammalian PFK interact with sites that have evolved from the duplicated phosphoenolpyruvate/ADP allosteric site of the ancestral PFK. The two sites for activators, fructose 2,6-bisphosphate and AMP or ADP, have evolved from the catalytic site of the ancestral precursor.     

Role of Ser530, Arg292, and His662 in the allosteric behavior of rabbit muscle phosphofructokinase.

Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is a potent allosteric activator of the ATP-dependent phosphofructokinase (PFK) in eukaryotes. Based on the sequence homology between rabbit muscle PFK and two bacterial PFKs and the crystal structures of the latter, Ser(530), Arg(292) and His(662) of the rabbit enzyme are implicated as binding sites for Fru-2,6-P(2). We report here the effects of three mutations, S530D, R292A, and H662A on the activation of rabbit muscle PFK by Fru-2,6-P(2). At pH 7.0 and the inhibitory concentrations of ATP, the native enzyme gives a classic sigmoidal response to changes in Fru-6-P concentration in the absence of Fru-2,6-P(2) and a nearly hyperbolic response in the presence of the activator. Under the same conditions, no activation was seen for S530D. On the other hand, H662A can be activated but requires a 10-fold or higher concentration of Fru-2,6-P(2). Limited activation was observed for mutant R292A. A model illustrating the sites for recognition of Fru-2,6-P(2) in rabbit muscle PFK as well as the mechanism of allosteric activation is proposed.     

The Crystal Structure of ATP-bound Phosphofructokinase from Trypanosoma brucei Reveals Conformational Transitions Different from those of Other Phosphofructokinases

The crystal structure of the ATP-bound form of the tetrameric phosphofructokinase (PFK) from Trypanosoma brucei enables detailed comparisons to be made with the structures of the apoenzyme form of the same enzyme, as well as with those of bacterial ATP-dependent and PP_i-dependent PFKs. The active site of T. brucei PFK (which is strictly ATP-dependent but belongs to the PP_i-dependent family by sequence similarities) is a chimera of the two types of PFK. In particular, the active site of T. brucei PFK possesses amino acid residues and structural features characteristic of both types of PFK. Conformational changes upon ATP binding are observed that include the opening of the active site to accommodate the two substrates, MgATP and fructose 6-phosphate, and a dramatic ordering of the C-terminal helices, which act like reaching arms to hold the tetramer together. These conformational transitions are fundamentally different from those of other ATP-dependent PFKs. The substantial differences in structure and mechanism of T. brucei PFK compared with bacterial and mammalian PFKs give optimism for the discovery of species-specific drugs for the treatment of diseases caused by protist parasites of the trypanosomatid family.     

Creation of an allosteric phosphofructokinase starting with a nonallosteric enzyme. The case of dictyostelium discoideum phosphofructokinase.

An allosteric phosphofructokinase (PFK) was created by sequence manipulation of the nonallosteric enzyme from the slime mold Dictyostelium discoideum (DdPFK). Most amino acid residues proposed as important for catalytic and allosteric sites are conserved in DdPFK except for a few of them, and their reversion did not modify its kinetic behavior. However, deletions at the unique C-terminal extension of this PFK produced a markedly allosteric enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited hysteresis in the time course, intense cooperativity (n(H) = 3.8), and a 200-fold decrease in the apparent affinity for fructose 6-phosphate (S(0.5) = 4500 microm), strong activation by fructose 2,6-bisphosphate (K(act) = 0.1 microm) and fructose 1,6-bisphosphate (K(act) = 40 microm), dependence on enzyme concentration, proton inhibition, and subunit association-dissociation in response to fructose 6-phosphate versus the nonhysteretic and hyperbolic wild-type enzyme (n(H) = 1.0; K(m) = 22 microm) that remained as a stable tetramer. Systematic deletions and point mutations at the C-tail region of DdPFK identified the last C-terminal residue, Leu(834), as critical to produce a nonallosteric enzyme. All allosteric mutants were practically insensitive to MgATP inhibition, suggesting that this effect does not involve the same allosteric transition as that responsible for fructose 6-phosphate cooperativity and fructose bisphosphate activation.     

6-Phosphofructo-1-kinase isoenzymes of the jejunal mucosa of rabbit, rat and mouse

1. 6-Phosphofructo-1-kinase (PFK) isoenzymes were studied in the jejunal mucosa of rabbit, rat and mouse. 2. The rat mucosal enzyme was found to be very similar to, although not identical with, the mouse mucosal enzyme, as the physical and regulatory properties of these two enzymes were nearly similar except that the immunological studies were dissimilar. 3. PFK prepared from rabbit mucosa showed different and distinct properties from the rat and mouse mucosal PFK when studied by (NH4)2SO4-precipitation, polyacrylamide gel electrophoresis, immunological cross-reactivity and regulatory properties. 4. The difference between the rabbit enzyme and the rat or mouse enzymes is suggested to be due to the lower rate of glycolysis observed in the rabbit jejunal mucosa as the total enzyme activities of the rabbit were found to be less than half of those activities of the rat and mouse mucosa. 5. The dissimilarities among the species in mucosal isoenzymes obtained in the present study are rather expected since the term isoenzyme is now properly reserved for forms that have been shown to be genetically distinct as shown for different tissues in the same species. Such multigenic control does not appear to have been established for the same tissue in different species.     

Comparison of pH-dependent allostery and dissociation for phosphofructokinases from Artemia embryos and rabbit muscle: nature of the enzymes acylated with diethylpyrocarbonate

Purified Artemia phosphofructokinase (PFK), unlike the rabbit skeletal muscle enzyme, displays allosteric kinetics at pH 8, a feature that is functionally significant since the intracellular pH of the developing brine shrimp embryo is greater than or equal to 7.9. Catalytic activity of the Artemia enzyme is severely suppressed by acidic pH even when assayed at the adenylate nucleotide concentrations existing in anaerobic embryos, which is consistent with the lack of a Pasteur effect in these organisms. For both PFK homologs, carbethoxylation reduces the sensitivity to ATP and citrate inhibition, the cooperativity as a function of fructose 6-phosphate concentration and the degree of activation in the presence ADP, AMP, and fructose 2,6-bisphosphate. Considering the role of histidine protonation in PFK allosteric control, the capacity for regulatory kinetics seen at pH 8 in the Artemia enzyme could be explained in part by upward shifts in pKa values of ionizable residues. pH-induced dissociation of tetrameric Artemia PFK into inactive subunits does not occur during catalytic inhibition at acidic pH (pH 6.5, 6 degrees C), as judged by 90 degree light scattering. This observation contrasts markedly with the dimerization and inactivation of rabbit PFK, but is shown not to be unique when compared to other selected PFK homologs. Neither the acute pH sensitivity of Artemia PFK nor the pH-induced hysteretic inactivation displayed by the rabbit enzyme are altered by carbethoxylation, suggesting that ionizable residues involved in these two processes are not the same ones involved in allosteric kinetics.     

Inositol 1,4-bisphosphate is an allosteric activator of muscle-type 6-phosphofructo-1-kinase.

The allosteric effects of various inositol biphosphate (InsP2) isomers and other inositol phosphates, of glycerophosphoinositol phosphates (GroPInsPx) and of phosphoinositides (PtdInsPx) on muscle-type 6-phosphofructo-1-kinase (PFK) were investigated. The binding of these substances to PFK was indirectly estimated by their ability to stabilize the tetrameric enzyme. At near-physiological concentrations of other allosteric effectors, muscle PFK was activated AMP-dependently by Ins(1,4)P2 (Ka = 43 microM), Ins(2,4)P2 (Ka = 70 microM) and GroPIns4P (Ka = 20 microM). These compounds activated PFK by a mechanism similar to that established for activating hexose bisphosphates. Indirect binding experiments indicated minimal Kd,app. values of about 5 microM for the binding of Ins(1,4)P2 in the presence of 0.1 mM-AMP at pH 7.4. This apparent affinity was comparable with that of fructose 1,6-bisphosphate and glucose 1,6-bisphosphate at identical conditions. The enzyme was also found to interact specifically with PtdIns4P (Kd,app. = 37 microM), the inositol phospholipid carrying Ins(1,4)P2 as its head group. The regulatory behaviour of muscle-type PFK in vitro and the concentrations of Ins(1,4)P2 in vivo (between 4 and greater than 50 nmol/g wet wt. of tissue) are consistent with the hypothesis that there is a functional interaction in vivo. Furthermore, a role of PtdIns4P in membrane compartmentation of PFK is suggested. Comparative experiments with liver PFK indicate that these regulatory properties may be relatively specific for the muscle isoform. Unlike muscle PFK, the liver isoform was slightly activated by sub-micromolar concentrations of Ins(1,4,5)P3.     

Activation of 6-phosphofructokinase via phosphorylation by Pkn4, a protein Ser/Thr kinase of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that exhibits a communal lifestyle during vegetative growth and multicellular development, forming fruiting bodies filled with spores. It contains at least 13 eukaryotic-like protein Ser/Thr kinases (PSTKs from Pkn1 to Pkn13). In the present report, we demonstrate that Pkn4, the gene located 18 bp downstream of the gene for 6-phosphofructokinase (PFK), is a PSTK for M. xanthus PFK (Mx-PFK), the key regulatory enzyme in glycolysis. Both Pkn4 and Mx-PFK were expressed in Escherichia coli and purified. Mx-PFK was found to be phosphorylated by Pkn4 at Thr-226, which is presumed to be located in the allosteric effector site of the PFK. The phosphorylation of Mx-PFK enhanced its activity 2.7-fold, indicating that Pkn4 plays an important role in glucose metabolism. Although PFKs from other organisms are known to be tetrameric enzymes, Mx-PFK is composed of an octamer and is dissociated to tetramers in the presence of phosphoenolpyruvate (PEP), an allosteric inhibitor for PFK. Furthermore, phosphorylation of PFK by Pkn4 is almost completely inhibited by PEP. Mx-PFK is associated with the regulatory domain of Pkn4, and this association is inhibited by PEP. This is the first demonstration that a prokaryotic PFK is regulated by phosphorylation by PSTK in prokaryotes.     

Phosphofructokinase from muscle of the marine giant barnacle Austromegabalanus psittacus: kinetic characterization and effect of in vitro phosphorylation

The kinetic properties of phosphofructokinase from muscle of the giant cirripede Austromegabalanus psittacus were characterized, after partial purification by ion exchange chromatography on DEAE-cellulose. This enzyme showed differences regarding PFKs from other marine invertebrates: the affinity for fructose 6-phosphate (Fru 6-P) was very low, with an S(0.5) of 22.6+/-1.4 mM (mean+/-S.D., n=3), and a high cooperativity (n(H) of 2.90+/-0.21; mean+/-S.D., n=3). The barnacle PFK showed hyperbolic saturation kinetics for ATP (apparent K(m ATP)=70 microM, at 5 mM Fru 6-P, in the presence of 2 mM ammonium sulfate). ATP concentrations higher than 1 mM inhibited the enzyme. Ammonium sulfate activated the PFK several folds, increasing the affinity of the enzyme for Fru 6-P and V(max). 5'-AMP (0.2 mM) increased the affinity for Fru 6-P (S(0.5) of 6.2 mM). Fructose 2,6-bisphosphate activated the PFK, with a maximal activation at concentrations higher than 2 microM. Citrate reverted the activation of PFK produced by 0.2 mM 5'-AMP (IC(50 citrate)=2.0 mM), producing a higher inhibition than that exerted on other invertebrate PFKs. Barnacle muscular PFK was activated in vitro after exposure to exogenous cyclic-AMP (0.1 mM) as well as by phosphatidylserine (50 microg/ml), indicating a possible control by protein kinase A and a phospholipid dependent protein kinase (PKC). The results suggest a highly regulated enzyme in vivo, by allosteric mechanisms and also by protein phosphorylation.     

Phosphofructokinase from the epithelial cells of rat small intestine. Comparison of regulatory properties with those of skeletal muscle, liver and brain phosphofructokinase

The regulatory properties of phosphofructokinase from rat mucosa, liver, brain and muscle were investigated. Mucosal phosphofructokinase displayed cooperativity with respect to fructose 6-phosphate at pH 7.0 and so did the muscle, brain and liver isoenzymes. All these four isoenzymes were inhibited by ATP, the mucosal isoenzyme being the least inhibited. They were also inhibited by citrate and creatine phosphate. AMP, ADP, glucose 1,6-diphosphate, fructose 2,6-bisphosphate and inorganic phosphate were all strong activators for the mucosal, brain, liver and muscle phosphofructokinase, but the mucosal isoenzyme was found to be more activated than the others, accounting for the higher rates of glycolysis observed in mucosa. The results suggest that mucosal phosphofructokinase is unique and different from all the other isoenzymes.     

Self-association of rabbit muscle phosphofructokinase: effects of ligands

The effects of ligands on the self-association of rabbit muscle phosphofructokinase (PFK) were investigated by velocity sedimentation at pH 7.0 and 23 degrees C. The concentration dependence of the weight-average sedimentation coefficient was monitored in the presence of these ligands. The mode of association and equilibrium constants characterizing each association step were determined by theoretical fitting of the sedimentation data. The simplest mode of association for the PFK system is M in equilibrium M2 equilibrium M4 in equilibrium M16. Ligands and temperature would perturb the various equilibrium constants without altering the mode of association. The apparent equilibrium constants for the formation of tetramer, K4app, are increased in the presence of 0.1 mM ATP and 1.0 mM fructose 6-phosphate. The value of the sedimentation coefficient for the tetramer, S4 degrees, that would best fit the data is 12.4 S instead of 13.5 S determined in the absence of substrates, thus implying a structural change in the tetramer induced by substrates. Only an insignificant amount of dimer is present under the experimental conditions. The presence of activators, ADP or phosphate, enhances the formation of tetramers, and S4 degrees assumes a value of 13.5 S. Similar results are obtained with decreasing concentrations of proton. The presence of the inhibitor, citrate, however, favors the formation of dimers. The equilibrium constants determined as a function of ADP concentration were further analyzed by the linked-function theory derived by Wyman [Wyman, J. (1964) Adv. Protein Chem. 19, 224--285], leading to the conclusion that the formation of a tetramer involves the binding of two additional molecules of ADP per monomer. Similar analysis results in a conclusion that the formation of a dimer involves the binding of one additional molecule of citrate per phosphofructokinase subunit.     

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum phosphofructokinase

The nucleotide sequence of a full-length cDNA encoding phosphofructokinase (PFK) enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2,653 bases comprises a single open reading frame of 2,452 bases and a noncoding region of 201 bases after the stop codon. The mature protein contains 812 amino acids and has a molecular mass of 90,900 Da. The amino acid sequences of several peptides derived from the purified protein show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the protein with those of 3 other worms as well as those of human, rabbit, and bacterial enzymes reveals highly conserved regions interrupted with stretches of lesser sequence similarity. Analyses of the subunit primary structure reveal, as in other eukaryotic PFKs, that the amino-terminal half is homologous to the carboxy-terminal half, supporting the hypothesis that the PFK gene evolved by duplication of the prokaryotic gene and that the allosteric sites arose by mutations at the catalytic site. The location of the phosphorylation site is unique and different compared with other PFKs and plays a key role in regulation of the enzyme activity. Structural motifs such as the putative substrate and effector binding domains and also the key amino acids involved therein are clearly identified by alignment of all the PFK protein sequences.