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PubMed1995

Functional expression of human mutant phosphofructokinase in yeast: genetic defects in French Canadian and Swiss patients with phosphofructokinase deficiency.

Human phosphofructokinase (PFK) is a tetrameric enzyme, encoded by muscle, liver, and platelet genes. Deficiency of muscle PFK (PFK-M), glycogenosis type VII (Tarui disease), is an autosomal recessive disorder characterized by an exertional myopathy and hemolytic syndrome. Several disease-causing mutations have been identified in the PFK-M gene in Japanese, Ashkenazi Jewish, and Italian patients. We describe the genetic defects in French Canadian and Swiss patients with the disease, and we use a genetically well-defined yeast system devoid of endogenous PFK for structure-function studies of the mutant PFKs. A G-to-A transition at codon 209-in exon 8 of the PFK-M gene, changing an encoded Gly to Asp, is responsible for the disease in a homozygous French Canadian patient. Gly-209-mutated protein is completely inactive in the yeast system. The Swiss patient is a genetic compound, carrying a G-to-A transition at codon 100 in exon 6 (Arg to Gln) and a G-to-A transition at codon 696 in exon 22 (Arg to His). The mutants expressed in yeast generate functional enzyme with modest changes in thermal stability. The advantages and limitations of the yeast system for expression of human mutant PFKs are discussed.

Adult↗
PubMed1998

The pyrophosphate-dependent phosphofructokinase of the protist, Trichomonas vaginalis, and the evolutionary relationships of protist phosphofructokinases.

The pyrophosphate-dependent phosphofructokinase (PPi-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5' half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PPi-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PPi-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PPi-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the alpha- and beta-subunits of plant PPi-PFKs. The third group ("X") containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group ("Y") comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PPi-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers.

Amino Acid Sequence↗
PubMed1983

Phosphofructokinase activities in photosynthetic organisms : the occurrence of pyrophosphate-dependent 6-phosphofructokinase in plants and algae.

A pyrophosphate-dependent phosphofructokinase (PPi-PFK) activity is detectable in extracts of a wide variety of primitive and advanced plants, the Charalean algae, and in the photosynthetic bacterium, Rhodospirillum rubrum. Angiosperms with extractable PPi-PFK activities 4- to 70-fold higher than the respective ATP-PFK activities tend to be succulent and to exhibit CAM. Even though PPi-PFK activity is not detected in crude extracts of some well known CAM plants, e.g. plants in the Crassulaceae, gel filtration of the extract and/or inclusion of the PPi-PFK activator, fructose 2,6-bisphosphate, in the assay reveals that a PPi-PFK activity is present in these species. Fructose 2,6-bisphosphate likewise activates PPi-PFK activities in extracts of C(3) and C(4) plants. C(3) and C(4) plant PPi-PFK activities are roughly equivalent to ATP-PFK activities in the same species. PPi-PFK activity is also detected in some bryophytes, lower vascular plants, ferns, and gymnosperms. The Charophytes, advanced algae presumed to be similar to species ancestral to vascular plants, exhibit at least 4-fold higher PPi-PFK than ATP-PFK activities. R. rubrum also exhibits a much higher PPi-PFK activity than ATP-PFK activity. These data indicate that PPi-PFK may serve as an alternate enzyme to ATP-PFK in glycolysis in a wide range of photosynthetic organisms.

Journal Article↗
PubMed1998

PPi-dependent phosphofructokinase from Thermoproteus tenax, an archaeal descendant of an ancient line in phosphofructokinase evolution.

Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PPi-dependent PFK (PPi-PFK; EC 2.7.1.90), which uses PPi instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PPi-PFK from the anaerobic, hyperthermophilic archaeon Thermoproteus tenax, purified the enzyme to homogeneity, and sequenced the gene. The approximately 100-kDa PPi-PFK from T. tenax consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PPi-PFKs from the genus Propionibacterium, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PPi-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PPi as a cosubstrate.

Amino Acid Sequence↗
PubMed1975

Catabolism of D-fructose and D-ribose by Pseudomonas doudoroffii. II. Properties of 1-phosphofructokinase and 6-phosphofructokinase.

1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudmonas doudoroffii were partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. The pH optima of these enzymes were 9.0 and 8.5, respectively. 2. When the concentrations of the substrates of the 1-PFK reaction were varied, Michaelis-Menten kinetics were observed. The Kms for D-fructose-1-P (F-1-P) and ATP were 3.03 X 10(-4) M and 3.39 X 10(-4) M, respectively. Variation of MgCl2 at fixed concentrations of F-1-P and ATP resulted in sigmoidal kinetics; about 10 mM MgCl2 was necessary for maximal activity. Activity of 1-PFK was inhibited when the ratio of ATP:Mg++ was higher than 0.5, suggesting that ATP:2Mg++ was the substrate and that free ATP was inhibitory. Although an absolute requirement for K+ or NH4+ could not be demonstrated, these cations stimulated the rate of the reaction. Activity of 1-PFK was not significantly affected by 3 mM AMP, cyclic-AMP, Pi, D-fructose-6-P (F-6-P), ADP, P-enolpyruvate (PEP), pyruvate, citrate, or L-gluamate. 3. Sigmoidal kinetics were observed for 6-PFK when the concentration of F-6-P was increased and the level of ATP was kept constant. Activity of 6-PFK was increased by ADP, inhibited by PEP, and unaffected by 3 mM AMP, cyclic-AMP, Pi, F-1-P, pyruvate, or citrate.

Adenosine Triphosphate↗
PubMed1990

Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the alpha- and beta-subunits and with the ATP-dependent phosphofructokinase.

Full-length cDNA clones for the alpha- and beta-subunits of pyrophosphate-fructose 6-phosphate 1-phosphotransferase have been isolated from a cDNA expression library derived from potato tuber poly(A)+ RNA. The nucleotide sequences indicate that the alpha- and beta-subunits are related with about 40% of amino acid residues being identical. A comparison of the deduced amino acid sequences of both subunits of this enzyme with that of the major ATP-dependent fructose 6-phosphate 1-phosphotransferase from Escherichia coli (Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994) showed little homology between the proteins except for regions involved in the binding of fructose 6-phosphate/fructose, 1,6-bisphosphate and possibly between regions binding pyrophosphate and the beta- and gamma-phosphates of ADP/ATP. A comparison of the derived secondary structures of the two subunits of the PPi-dependent enzyme with the known secondary structure of the E. coli ATP-dependent enzyme indicated that the overall structure of these enzymes is similar. These data suggest that catalytic activity resides on the beta-subunit of the pyrophosphate-dependent enzyme.

Adenosine Triphosphate↗
PubMed1982

An alteration in phosphofructokinase 2 of Escherichia coli which impairs gluconeogenic growth and improves growth on sugars.

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.

Carbohydrates↗
PubMed1987

Phosphofructokinase from Fasciola hepatica: activation by phosphorylation and other regulatory properties distinct from the mammalian enzyme.

Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for phosphorylation and Fru-2,6-P2 in regulation of fluke phosphofructokinase.

Adenosine Triphosphate↗
PubMed1976

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.

Adenosine Triphosphate↗
PubMed1988

Liver (B-type) phosphofructokinase mRNA. Cloning, structure, and expression.

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.

Amino Acid Sequence↗
PubMed1985

Isolation and characterization of phosphofructokinase C from rabbit brain.

Phosphofructokinase from rabbit brain consists of hybrids of the A, B, and C isozymes. Phosphofructokinase C was isolated from a purified mixture of such hybrids in a 2-step procedure. In the first step, phosphofructokinase B was removed by chromatography on DEAE-Sephadex. In the second step, subunits of phosphofructokinases A and C were separated by dissociation at pH 5.0 followed by chromatography on carboxymethylcellulose. The separated isozymes were then reassociated by neutralization. Phosphofructokinase C was structurally distinct from phosphofructokinases A (obtained from muscle or brain) and B (obtained from liver) as shown by one-dimensional chymotryptic and staphylococcal V8 protease fingerprints of all three isozymes. In addition, phosphofructokinase C cross-reacted weakly or not at all with antisera raised against phosphofructokinase B or phosphofructokinase A. Phosphofructokinase C was also kinetically distinct from the A and B isozymes. The C isozyme was more sensitive than the A isozyme but less sensitive than the B isozyme to inhibition by ATP, was less sensitive than the A isozyme but more sensitive than the B isozyme to inhibition by citrate, and was less sensitive than either of the other two isozymes to activation by inorganic phosphate, AMP, and fructose 2,6-bisphosphate. The self-association properties of phosphofructokinase C differed from those of the A and B isozymes in that at pH 8.0, the C isozyme did not form oligomers larger than a tetramer under conditions where the other two isozymes did. Thus the properties of phosphofructokinase C are in general quite distinct from those of the other two phosphofructokinase isozymes.

Adenosine Monophosphate↗
PubMed1981

Developmental changes in heart and muscle phosphofructokinase isozymes.

Phosphofructokinase isozymes of fetal, neonatal, and adult rat heart and skeletal muscle were characterized by DEAE-cellulose chromatography, agarose gel electrophoresis, and immunodiffusion with specific antisera. The results of these studies indicate that in skeletal muscle and heart the levels of the major liver phosphofructokinase isozyme (PFK-L2) and the muscle phosphofructokinase isozyme (PFK-M) are dependent on the developmental status of the rat. For example, PFK-L2 and PFK-M are present in fetal and early neonatal skeletal muscle; whereas in adult skeletal muscle, only PFK-M is detectable. By DEAE- cellulose chromatography, PFK-L2 activity was estimated to be 2.4 units/g (41% of total phosphofructokinase activity) in fetal muscle, very low and not resolved from PFK-M in 7-day neonatal muscle, and not detectable in adult muscle. Further, PFK-M activity was found to be 3.4 units/g (59% of total phosphofructokinase activity), 10 units/g, and 31.6 units/g in fetal, 7-day neonatal, and adult skeletal muscle, respectively. The developmental changes of heart phosphofructokinase isozymes differ considerably from that of the skeletal muscle phosphofructokinase isozymes. In fetal heart, PFK-L2 is the major phosphofructokinase isozyme (5.6 units/g), constituting 67% of total phosphofructokinase activity. Further, in fetal heart another phosphofructokinase isozyme (33% of total phosphofructokinase activity) was found by DEAE-cellulose chromatography which is different from PFK-M and PFK-L2. In 7-day neonatal and adult heart, PFK-M and PFK-L2 are the only detectable phosphofructokinase isozymes. Varying from 5.6 units/g (44% of total) in 7-day neonatal to 5.9 units/g (40% of total) in adult heart, PFK-L2 activity remains fairly constant. Also, PFK-M is very low in fetal heart but increases within 1 week postpartum to 5.5 units/g (50% of total activity) and to 8.9 units/g (60% of total activity) in adult heart.

Aging↗