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A 6-phosphofructo-1-kinase-coding gene (pfk) has been purified from a human genomic library cloned in the lambda EMBL4 phage vector. This clone contains the nontranslated 5' flanking region of the human muscle pfk gene. Comparison of the nucleotide sequence determined by us with that of the human muscle pfk cDNA [Nakajima et al., FEBS Lett. 223 (1987) 113-116] indicates the presence of an additional intron extending from nucleotide (nt) -97 to -9 upstream of the ATG start codon. Furthermore, the human muscle pfk gene is more AT-rich than the rabbit gene. The available sequence of the two cDNAs shows 256 nt differences. Surprisingly, 71% of these sites are A's and T's in the human cDNA and C's and G's in the rabbit gene.

Phosphofructokinase (PFK;EC 2.7.1.11) activity in Drosophila melanogaster is controlled by a single dosage-sensitive region of the genome between 45F and 47E of chromosome IIR. Only a single form of PFK was detected electrophoretically in both adults and larvae. Nearly 90% of the PFK activity in adults is localized to the thorax. Purification of the enzyme was hampered by the extreme lability of Drosophila PFK; however, a 36-fold partial purification was achieved.

Mouse liver mRNA enriched in sequences coding for liver phosphofructokinase by polysome immunoadsorption was used as a template for the synthesis of cDNA. The double-stranded cDNA was inserted into the expression vector lambda gt11 and cloned. Preliminary identification of clones containing cDNA sequences for phosphofructokinase was made by screening the library with anti-rat liver phosphofructokinase serum and horseradish peroxidase-conjugated goat anti-rabbit IgG as second antibody. Subsequently, by selecting antibodies specific to fusion proteins expressed by putative clones and by reacting with Western blots of mouse liver proteins several clones were positively identified as containing liver phosphofructokinase sequences. A cDNA clone corresponding to 2708 nucleotides of liver phosphofructokinase mRNA was further characterized and sequenced. The liver phosphofructokinase mRNA has an open reading frame of 2343 nucleotides followed by a 3'-untranslated region of 303 nucleotides. The G/C-rich (76%) portion of the 5'-untranslated region precedes a characteristic translational start site of CCGCC(AUG). The mRNA coding sequence indicates that the liver phosphofructokinase subunit is composed of 780 amino acid residues and has a Mr of 85,000. Comparison of the deduced amino acid sequence of mouse liver phosphofructokinase with the known rabbit muscle phosphofructokinase shows 68% homology. The N-half of the liver phosphofructokinase has conserved substrate binding sites for ATP and fructose-6-P. The 25 C-terminal residues, which contain the ATP inhibitory site, are the least homologous (20%) but contain a putative phosphorylation site (Arg-Arg-X-X-Ser). The liver phosphofructokinase mRNA is under nutritional and hormonal regulation. The liver phosphofructokinase mRNA level increased 4-fold when previously starved mice were refed a high carbohydrate, fat-free diet. This increase in mRNA level was blocked by 50% by the administration of dibutyryl cAMP. The induction of liver phosphofructokinase mRNA by fasting/refeeding was also diminished in streptozotocin diabetic mice.

Although several of the genes mapped to human chromosome 21 have been assigned to mouse chromosome 16, it has not yet been possible to do this for the gene for the phosphofructokinase liver type subunit (PFKL). The goal of this study was to determine if there is a 1.5-fold increase of the PFK activity in fetal trisomy 16 mice, which, if present, would be indicative of a gene dosage effect. However, rather than an increase, an almost 100% decrease of the mean PFK activity was observed in fetal trisomy 16 liver at both 14 and 17 days of gestation when compared to littermate controls. This is the first biochemical abnormality detected in trisomy 16 fetal liver. In contrast, no significant differences in the mean PFK activity in homogenized whole fetus or brain trisomy 16 and diploid controls were observed. A developmental maturational effect of the PFK activity was observed in fetal liver from normal diploid mice, with a 3-fold increase of the activity from day 14 to day 18 of gestation and a further 2-fold increase to adulthood. The decreased PFK activity in fetal trisomy 16 liver may therefore be the result of delayed maturation of the liver in trisomic fetuses.

The aggregation states of Escherichia coli phosphofructokinase 2 (Pfk-2) and of a mutant enzyme (Pfk-2*) altered in the inhibitory allosteric site for MgATP were measured in the presence and in the absence of substrates and products of the reaction. When sucrose gradient ultracentrifugation experiments were performed in the absence of added ligands, both enzymes sedimented as dimers. Likewise, at low concentrations of both substrates (0.1 mM) the aggregation state of Pfk-2 and Pfk-2* corresponded to a dimer. However, in the presence of 1 mM MgATP alone, Pfk-2 sedimented as a tetramer, whereas Pfk-2* sedimented as a dimer. At a low fructose 6-phosphate concentration (0.1 mM) and an inhibitory concentration of MgATP (4 mM), Pfk-2 sedimented as a tetramer. However, at the same MgATP concentration but at a higher fructose-6-P concentration (1 mM), a condition under which Pfk-2 is not inhibited by the Mg-nucleotide complex, the enzyme sedimented as a dimer. Pfk-2* is not inhibited under these conditions and sedimented as a dimer in each case. Thus, the effectiveness of MgATP in promoting the aggregation of Pfk-2 and Pfk-2* parallels the inhibitability of the enzymes by the nucleotide complex. However, ATP4-, a potent inhibitor of Pfk-2 and Pfk-2* that binds to the catalytic site of the enzymes, had no effect upon their aggregation states. Possibly Pfk-2* is not able to form a tetramer because of an alteration in the regulatory site for the Mg-nucleotide complex.

Eight different enzymes for glycolysis and alcoholic fermentation were overproduced in a common Saccharomyces cerevisiae strain by placing their genes on multicopy vectors. The specific enzyme activities were increased between 3.7- and 13.9-fold above the wild-type level. The overproduction of the different glycolytic enzymes had no effect on the rate of ethanol formation, even with those enzymes that catalyse irreversible steps: hexokinase, phosphofructokinase and pyruvate kinase. Also the simultaneous increase in the activities of pairs of enzymes such as pyruvate kinase and phosphofructokinase or pyruvate decarboxylase and alcohol dehydrogenase, did not increase the rate of ethanol production. The levels of key glycolytic metabolites were also normal, compared to the reference strain.

Many enzymes are subject to allosteric control, often with inhibitors and activators binding to the same effector site. Phosphofructokinase in Escherichia coli is such an enzyme, being inhibited by phosphoenolpyruvate (PEP) and activated by ADP and GDP. How do individual interactions with effectors affect the balance between activation and inhibition, especially when both ligands share aspects of the same binding site? We find that mutation of a single residue in the effector site, Glu----Ala 187, leads to PEP being an activator rather than an inhibitor. With low concentrations of the substrate fructose-6-phosphate, the mutant enzyme is more than one hundred times more active than wild-type enzyme at millimolar concentrations of PEP. The classical Monod-Wyman-Changeux two-state model is too simple to account for the properties of the mutant enzyme.

The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* was measured over a wide range of Mg2+ and ATP concentrations. MgATP2- inhibited only the Pfk-2 enzyme, with a degree of cooperativity of 1.5. This inhibition was relieved upon increasing the fructose-6-P concentration or by lowering the pH of the reaction mixture. Other nucleotides used as phosphate donors instead of ATP did not inhibit. MgATP2- was the true substrate for both enzymes and their Km values for this compound were not affected by an increase of the free Mg2+ concentration. However, free Mg2+ partially relieved the MgATP2- inhibition of Pfk-2 under conditions where the ATP4- concentration was negligible, without changes in the degree of cooperativity. ATP4- acted as a strong competitive inhibitor of both Pfk-2 and Pfk-2* with respect to MgATP2- with Ki values of 10 and 8 microM, respectively. ADP, AMP, and cAMP did not prevent the MgATP2- inhibition of Pfk-2. These results suggest the presence of an allosteric site for MgATP2- in Pfk-2 responsible for the MgATP2- inhibition, which is altered in Pfk-2* as a consequence of the structural mutation.

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3'-untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5'-untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C- and N-halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).

Phosphofructokinase (PFK) isozymes and their physiological significance have been the focus of extensive research. The majority of this work has been centered around the PFK isozymes of rat, human and rabbit tissues. Consequently, this review emphasizes these studies. Additionally, a review of PFK isozymes in chickens, mice, guinea pig, and pig is presented. The relationship of the properties of each PFK isozyme in different tissues to the rates of glycolysis and/or gluconeogenesis in those tissues is discussed where possible. Moreover, the contribution of the different PFK isoenzymes to alterations of the glycolytic rate in various tissues is discussed in relationship to variations in nutritional, hormonal, developmental or pathological status of the animal.

The main phosphofructokinase of Escherichia coli (PFK I) is an extensively studied allosteric enzyme specified by the pfkA gene. A nonallosteric phosphofructokinase was reported (Fraenkel, D.G., Kotlarz, D., and Bluc, H. (1973) J. Biol. Chem. 248, 4865-4866) in strains carrying the pfkB1 mutation, a suppressor of pfkA mutants, and very low levels of this enzyme have also been detected in strains not carrying the suppressor (i.e. pfkB+). The nonallosteric protein has now been prepared pure from three strains, one carrying pfkB1 and pfkA+, one carrying pfkB1 and completely deleted for pfkA, and one carrying pfkB+ and also deleted for pfkA. It is apparently the same enzyme (PFK II) in all three strains, which shows that pfkB1 is a mutation affecting the amount of a normally minor isozyme. PFK II is a tetramer of slightly larger subunit molecular weight than PFK I (36,000 and 34,000, respectively). No immunological cross-reactivity was detected between PFK II and PFK I. Unlike PFK I, PFK II does not show cooperative interactions with fructose-6-P, inhibition by P-enolpyruvate, or activation by ADP. Also unlike PFK I, PFK II is somewhat sensitive to inhibition by fructose-1,6-P2 and can use tagatose-6-P as substrate. Both enzymes can perform the reverse reaction, fructose-6-P + ATP from fructose-1,6-P2 + ADP in vitro, but not in vivo. The normal function of PFK II is not known.