pyrophosphate and fructose-1-6-diphosphate

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

The energy derived from pyrophosphate (PPi) hydrolysis is used to pump protons across the tonoplast membrane, thus forming a proton gradient. In a plant's cytosol, the concentration of PPi varies between 10 and 800 microm, and the PPi concentration needed for one-half maximal activity of the maize (Zea mays) root tonoplast H+-pyrophosphatase is 30 microm. In this report, we show that the H+-pyrophosphatase of maize root vacuoles is able to hydrolyze PPi (Reaction 2) formed by Reaction 1, which is catalyzed by PPi-dependent phosphofructokinase (PFP): Fructose-1,6-bisphosphate (F1,6BP) + Pi <--> PPi +Fructose-6-phosphate (F6 P) (reaction 1) PPi --> 2 Pi (reaction 2) H+cyt --> H+vac (reaction 3) F1,6BP + H+cyt <--> H+vac + F6P + Pi (reaction 4) During the steady state, one-half of the inorganic phosphate released (Reaction 4) is ultimately derived from F1,6BP, whereas PFP continuously regenerates the pyrophosphate (PPi) hydrolyzed. A proton gradient (DeltapH) can be built up in tonoplast vesicles using PFP as a PPi-regenerating system. The Delta pH formed by the H+-pyrophosphatase can be dissipated by addition of 20 mm F6P, which drives Reaction 1 to the left and decreases the PPi available for the H+-pyrophosphatase. The maximal Delta pH attained by the pyrophosphatase coupled to the PFP reaction can be maintained by PFP activities far below those found in higher plants tissues.

Topics: Biological Transport; Diphosphates; Fabaceae; Fructosediphosphates; Kinetics; Phosphofructokinase-2; Protons; Zea mays

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

A PPi-dependent phosphofructotransferase (PPi-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1, 6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430-fold). PPi was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or Pi could not substitute for PPi. The PPi-dependent reaction (2.0 mM PPi) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (less than or equal to 300 microM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1, 6-P2 to F-6-P, required Pi; arsenate could substitute for Pi, but not PPi or any other nucleotide tested. The computer-derived kinetic constants (+/- standard deviation) for the reaction in the PPi-driven direction of F-1, 6-P2 were as follows: v, 38.9 +/- 0.48 mM min-1; Ka(PPi), 0.11 +/- 0.04 mM; Kb(F-6-P), 0.65 +/- 0.15 mM; and Kia(PPi), 0.39 +/- 0.11 mM. A. laidlawii B-PG9 required PPi not only for the PPi-phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity. a dependency unknown in any other organism. In A. laidlawii B-PG9, the PPi requirement may be met by reactions in this organism already known to synthesize PPi (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PPi instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process.

Topics: Acholeplasma laidlawii; Diphosphates; Fructosediphosphates; Hydrogen-Ion Concentration; Kinetics; Magnesium; Molecular Weight; Phosphates; Phosphotransferases

The lactic dehydrogenase of a strain of Streptococcus bovis specifically requires fructose-1,6-diphosphate for activity. Phosphate or fructose-1-6-diphosphate prevents inactivation of the dehydrogenase, but phosphate and other compounds cannot be substituted for the fructose-1,6-diphosphate required for activity. Lactic dehydrogenases of other species of Streptococcus show a similar requirement for fructose-1,6-diphosphate.

Topics: Diphosphates; Fructose; Fructosediphosphates; Hexosephosphates; L-Lactate Dehydrogenase; Oxidoreductases; Phosphates; Research; Streptococcus

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

Topics: Diphosphates; Fructose; Fructosediphosphates; Hexosephosphates; Pregnancy

Topics: Carbohydrates; Colorimetry; Diphosphates; Fructose; Fructosediphosphates; Phosphates; Trioses

Topics: Creatine; Creatinine; Diphosphates; Fructose; Fructosediphosphates; Glycolysis; Hexosephosphates; Humans; Muscular Diseases; Muscular Dystrophies; Urinalysis

Topics: Diphosphates; Fructose; Fructosediphosphates; Heptoses; Hexosephosphates; Phosphoric Monoester Hydrolases; Photosynthesis

Topics: Diphosphates; Female; Fructose; Fructosediphosphates; Guinea Pigs; Hexosephosphates; Humans; Ions; Sodium; Sodium, Dietary; Uterus

Topics: Acetates; Acetic Acid; Acetobacter; Diphosphates; Fructose; Fructosediphosphates; Glycerophosphates; Hexosephosphates; Humans; Lipid Metabolism

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

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