pyrophosphate and fructose-6-phosphate

Recent evidence indicates that in as diverse organisms as unicellular eukaryotes, higher plants and prokaryotes, anaerobic glycolysis relies on a pyrophosphate-dependent phosphofructokinase instead of the classical ATP-dependent enzyme. This difference in phosphoryl donor specificity does not necessarily reflect a primitive metabolism, as thought earlier, but could rather be the result of convergent evolution, fostered by the energetic advantage conferred to the cell when glycolysis is the sole source of ATP.

Topics: Adenosine Triphosphate; Anaerobiosis; Biological Evolution; Diphosphates; Eukaryotic Cells; Fructosephosphates; Glycolysis; Phosphotransferases; Phylogeny; Plants; Prokaryotic Cells

This work reports the molecular cloning and heterologous expression of the genes coding for α and β subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg

Topics: Citrus sinensis; Cloning, Molecular; Diphosphates; Fructosediphosphates; Fructosephosphates; Gene Expression; Kinetics; Multiprotein Complexes; Phosphofructokinases; Phosphorylation; Phosphotransferases; Plant Proteins; Recombinant Proteins

Pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) was obtained as His₆-tagged protein by cloning of the pfp gene from the aerobic obligate methanotroph Methylomicrobium alcaliphilum 20Z and characterized. The recombinant PPi-PFK (4×45 kDa) was highly active, non-allosteric and stringently specific to pyrophosphate as the phosphoryl donor. The enzyme was more specific for the reverse reaction substrate fructose-1,6-bisphosphate (K(m) 0.095 mM, V(max) 805 U/mg of protein) than for the forward reaction substrate fructose-6-phosphate (K(m) 0.64 mM, V(max) 577 U/mg of protein). It also phosphorylated sedoheptulose-7-phosphate with much lower efficiency (K(m) 1.01 mM, V(max) 0.118 U/mg of protein). The kinetic properties of the M. alcaliphilum PP(i)-PFK were analyzed and compared with those of PP(i)-PFKs from other methanotrophs. The PP(i)-PFK from M. alcaliphilum shows highest sequence identity to PPi-PFK from obligate mesophilic methanotroph Methylomonas methanica (89%), and only low identity to the enzyme from thermotolerant Methylococcus capsulatus Bath (16%). This extensive sequence divergence of PPi-PFKs correlated with differential ability to phosphorylate sedoheptulose-7-phosphate and with the metabolic patterns of these bacteria assimilating C₁ substrate either via the ribulose monophoshate (RuMP) cycle or simultaneously via the RuMP and the Calvin cycles. Based on enzymic and genomic data, the involvement of PPi-PFK in pyrophosphate-dependent glycolysis in M. alcaliphilum 20Z was fist proposed.

Topics: Cluster Analysis; Diphosphates; Enzyme Activators; Fructosediphosphates; Fructosephosphates; Kinetics; Methylococcaceae; Molecular Weight; Phosphofructokinase-1; Phylogeny; Protein Multimerization; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Sugar Phosphates

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/alpha-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.

Topics: 4-Aminobenzoic Acid; Bacillus anthracis; Catalysis; Dihydropteroate Synthase; Diphosphates; Drug Resistance, Bacterial; Fructose-Bisphosphate Aldolase; Fructosephosphates; Glycerolphosphate Dehydrogenase; Kinetics; Magnesium Compounds; Models, Molecular; Molecular Structure; NAD; Phosphoric Acids; Phosphotransferases; Pterins; Recombinant Proteins; Sulfonamides; Triose-Phosphate Isomerase

The structure of the 60 kDa pyrophosphate (PP(i))-dependent phosphofructokinase (PFK) from Borrelia burgdorferi has been solved and refined (R(free) = 0.243) at 2.55 A resolution. The domain structure of eubacterial ATP-dependent PFKs is conserved in B. burgdorferi PFK, and there are three large insertions relative to E. coli PFK, including a helical domain containing a hairpin structure that interacts with the active site. Asp177, conserved in all PP(i) PFKs, negates the binding of the alpha-phosphate group of ATP and likely contacts the essential Mg(2+) cation via a water molecule. Asn181 blocks the binding of the adenine moiety of ATP. Lys203 hydrogen bonds to a sulfate anion that likely mimics PP(i) substrate binding.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Borrelia burgdorferi; Diphosphates; Escherichia coli Proteins; Fructosephosphates; Humans; Lyme Disease; Models, Molecular; Molecular Sequence Data; Molecular Structure; Phosphofructokinases; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Alignment

A pyrophosphate-dependent phosphofructokinase (PP(i)-PFK) and an ATP-dependent phosphofructokinase (ATP-PFK) from Thermotoga maritima have been cloned and characterized. The PP(i)-PFK is unique in that the K(m) and V(max) values indicate that polyphosphate is the preferred substrate over pyrophosphate; the enzyme in reality is a polyphosphate-dependent PFK. The ATP-PFK was not significantly affected by common allosteric effectors (e.g., phosphoenolpyruvate) but was strongly inhibited by PP(i) and polyphosphate. The results suggest that the control of the Embden-Meyerhof pathway in this organism is likely to be modulated by pyrophosphate and/or polyphosphate.

Topics: Adenosine Triphosphate; Allosteric Regulation; Diphosphates; Fructosephosphates; Phosphofructokinase-1; Phosphotransferases; Recombinant Proteins; Thermotoga maritima

The role of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) in developing leaves was studied using wild-type tobacco (Nicotiana tabacum L.) and transformants with decreased expression of PFP. (i) The leaf base, which is the youngest and most actively growing area of the leaf, had 2.5-fold higher PFP activity than the leaf tip. T3 transformants, with a 56-95% decrease in PFP activity in the leaf base and an 87-97% decrease in PFP activity in the leaf tip, were obtained by selfing and re-selfing individuals from two independent transformant lines. (ii) Other enzyme activities also showed a gradient from the leaf base to the leaf tip. There was a decrease in PFK and an increase in fructose-6-phosphate,2-kinase and plastidic fructose-1, 6-bisphosphatase, whereas cytosolic fructose-1,6-bisphosphatase activity was constant. None of these gradients was altered in the transformants. (iii) Fructose-2,6-bisphosphate (Fru2,6bisP) levels were similar at the base and tip of wild-type leaves in the dark. Illumination lead to a decrease in Fru2,6bisP at the leaf tip and an increase in Fru2,6bisP at the leaf base. Compared to wild-type plants, transformants with decreased expression of PFP had up to 2-fold higher Fru2,6bisP at the leaf tip in the dark, similar levels at the leaf tip in the light, 15-fold higher levels at the leaf base in the dark, and up to 4-fold higher levels at the leaf base in the light. (iv) To investigate metabolic fluxes, leaf discs were supplied with 14CO2 in the light or [14C]glucose in the light or the dark. Discs from the leaf tip had higher rates of photosynthesis than discs from the leaf base, whereas the rate of glucose uptake and metabolism was similar in both tissues. Significantly less label was incorporated into neutral sugars, and more into anionic compounds, cell wall and protein, and amino acids in discs from the leaf base. Metabolism of 14CO2 and [14C]glucose in transformants with low PFP was similar to that in wild-type plants, except that synthesis of neutral sugars from 14CO2 was slightly reduced in discs from the base of the leaf. (v) These results reveal that the role of PFP in the growing cells in the base of the leaf differs from that in mature leaf tissue. The increase in Fru2,6bisP in the light and the high activity of PFP relative to cytosolic fructose-1,6-bisphosphatase in the base of the leaf implicate PFP in the synthesis of sucrose in the light, as well as in glycolysis. The large increase in Fru2,6bisP a

Topics: Biological Transport; Carbohydrate Metabolism; Carbon Dioxide; Darkness; Diphosphates; Fructosediphosphates; Fructosephosphates; Gene Expression Regulation, Enzymologic; Glucose; Isotope Labeling; Light; Nicotiana; Phosphotransferases; Photosynthesis; Plant Leaves; Plants, Genetically Modified; Time Factors

Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. A phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the change in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary history, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Binding Sites; Cloning, Molecular; Diphosphates; Evolution, Molecular; Fructosephosphates; Inorganic Pyrophosphatase; Microbodies; Molecular Sequence Data; Phosphofructokinase-1; Phylogeny; Protein Binding; Protozoan Proteins; Pyrophosphatases; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Structure-Activity Relationship; Trypanosoma brucei brucei

Topics: Animals; Cell Line; Diphosphates; Fructosephosphates; Glucose-6-Phosphatase; Glucose-6-Phosphate; Glucosephosphates; Liver; Liver Neoplasms, Experimental; Rats; Substrate Specificity; Tumor Cells, Cultured

The steady-state kinetics of the reaction catalyzed by inorganic-pyrophosphate-dependent D-fructose-6-phosphate 1-phosphotransferase from Giardia lamblia have been investigated. The reactants for the forward and reverse reactions were the Mg-chelated complexes of pyrophosphate (PPi) and Pi. Uncomplexed ligands were not substrates. In the direction of phosphorylation of fructose-6-phosphate (F6P), initial velocity double-reciprocal plots for both PPi and F6P were intersecting suggesting sequential addition of substrates. Similarly, intersecting patterns were observed in the reverse reaction with either Pi or fructose-1,6-bisphosphate (FBP) as the variable substrate. Although the catalytic constants for the forward and reverse reactions were found to be identical (83 s-1), the kcat/Km for PPi is about two orders of magnitude higher than the kcat/Km for Pi, indicating that PPi is utilized much more efficiently than Pi. Product inhibition of Pi is competitive vs. PPi and noncompetitive vs. F6P, when the fixed substrate is subsaturating. Product inhibition by FBP was found to be noncompetitive with either Pi or F6P as the variable substrate. These results are consistent with a sequential ordered Bi Bi mechanism with PPi adding first and Pi dissociating last. In the reverse reaction, however, PPi and F6P were found to be noncompetitive with either Pi or FBP. Dead-end inhibition analysis with fructose 2,6-bisphosphate, a competitive substrate analog of FBP, gave uncompetitive inhibition with respect to Pi, indicating that fructose 2,6-bisphosphate (and hence FBP) binds after Pi. This kinetic mechanism is different from that observed with the enzyme from Propionibacterium freudenreichii, Entamoeba histolytica or Mung bean, which were concluded to be rapid equilibrium random mechanism.

Topics: Animals; Binding, Competitive; Chelating Agents; Diphosphates; Fructosediphosphates; Fructosephosphates; Giardia lamblia; Kinetics; Magnesium; Phosphotransferases; Substrate Specificity

Guard cells and palisade cells were dissected from freeze-dried leaflets of the broad bean, Vicia faba L. Individual cell samples (6-12 ng) were assayed for ATP-dependent and pyrophosphate-dependent phosphofructokinases. The assay indicator, NADH loss, was monitored in real time in oil droplets with a computer-driven microfluorometer. On a protein basis, both activities were 10-fold higher in guard cells than in palisade cells, indicating (i) elevated carbon metabolism in guard cells to meet demands for energy and carbon skeletons required during stomatal opening, and (ii) parallel glycolytic pathways in guard cells, one responsive to the potent regulatory metabolite fructose 2,6-bisphosphate and the other not. Future work will be devoted to clarifying the roles of the cytosolic and chloroplastic compartments in guard cells.

Topics: Adenosine Triphosphate; Diphosphates; Fabaceae; Fructosephosphates; Glycolysis; NAD; Phosphofructokinase-1; Phosphorylation; Plant Cells; Plants; Plants, Medicinal

Inorganic pyrophosphate (PPi) measurement in urine and synovial fluid has been established using the PPi-dependent phosphorylation of fructose-6-phosphate and subsequent reduction of dihydroxyacetone phosphate by NADH. The assay is linear up to 200 mumol/L, easy to perform and gives results comparable to more complex methods. Daily urinary output of PPi was independently related to both age (P = 0.0014) and sex (P = 0.0002). Men had higher values than women and older individuals excreted greater amounts. Male stone formers, younger than 45 years, had lower values than age matched male controls (P = 0.012). Younger female stone formers also tended to have lower values. In stone formers' urine significant and independent correlations were found of PPi excretion with urine volume (P = 0.004) and with phosphate excretion (P = 0.008). Oxalate excretion and that of other urine constituents and the degree of supersaturation with common stone-forming salts were not correlated with PPi. PPi excretion was markedly elevated in the urine of two patients with hypophosphatasia. The PPi concentration in synovial fluid from painful, swollen knee joints was elevated, but unrelated to the presence or absence of PPi or urate crystals.

Topics: Adolescent; Adult; Aged; Aging; Analysis of Variance; Child; Child, Preschool; Dihydroxyacetone Phosphate; Diphosphates; Female; Fructosephosphates; Humans; Hypophosphatasia; Kidney Calculi; Knee Joint; Male; Middle Aged; NAD; Oxidation-Reduction; Phosphorylation; Risk Factors; Sex Factors; Synovial Fluid

The pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii is rapidly inactivated by low concentrations of pyridoxal 5'-phosphate (PLP). The inactivation is first order with respect to PLP and the rate increases linearly with PLP concentrations suggesting that over the concentration range used no significant E-PLP complex accumulates during inactivation. The rate of inactivation decreases at high and low pH and this is discussed in terms of the mechanism of Schiff base formation. The presence of any reactants decreases the rate of inactivation to 0 at infinite concentration. This protection against inactivation has been used to obtain the pH dependence of the dissociation constants of all enzyme-reactant binary complexes. Reduction of the PLP-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 6-phosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphate probably directly coordinated to these phosphates.

Topics: Algorithms; Diphosphates; Fructosephosphates; Hydrogen-Ion Concentration; Kinetics; Lysine; Phosphofructokinase-1; Propionibacterium; Pyridoxal Phosphate

6-Phosphofructokinase (6-PFK) activities have been measured in cell extracts from a number of Bacteroides species. Two main types of 6-PFK were found: an ATP-linked 6-PFK and a PPi-linked 6-PFK. In most strains both of these activities were found, although in two strains only ATP-linked 6-PFK was present. The PPi-linked 6-PFK activity was always higher, when both activities were present, and showed Michaelis-Menten kinetics with respect to fructose 6-phosphate. In contrast, the ATP-linked 6-PFK activity usually gave sigmoid kinetics with respect to fructose 6-phosphate, although several strains were found to have activities with Michaelis-Menten kinetics. Conditions for measuring maximum activities of ATP-linked 6-PFK were not identical for different strains, and the activities were rather unstable in extracts. The possible consequences of the observation that most Bacteroides strains possess both an ATP-linked and a PPi-linked 6-PFK are discussed.

Topics: Adenosine Triphosphate; Bacteroides; Bacteroides fragilis; Diphosphates; Fructosephosphates; Kinetics; Phosphofructokinase-1; Phosphorylation; Prevotella melaninogenica

Topics: Colorimetry; Diphosphates; Fructose; Fructosediphosphates; Fructosephosphates; Hexosephosphates; Humans

Topics: Diphosphates; Escherichia coli; Fructose; Fructosediphosphates; Fructosephosphates