pyrophosphate and fructose-2-6-diphosphate

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

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

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

PPi-dependent phosphofructokinase (PPi-PFK) was detected in extracts of the amoeba Naegleria fowleri, with a specific activity of about 15-30 nmol/min per mg of protein, which was increased about 2-fold by 0.5 mM AMP. PPi-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30 degrees C in the presence of AMP. N. fowleri PPi-PFK was purified more than 1100-fold to near homogeneity with a yield of about 25%. The pure enzyme had a specific activity of 65 mumol/min per mg of protein, and SDS/PAGE analysis showed a single band, of 51 kDa. Size-exclusion chromatography revealed the existence of two forms: a large one (approximately 180 kDa), presumably a tetramer, which was active, and a smaller one (approximately 45 kDa), presumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30 degrees C in the presence of 0.5 mM AMP. Reactivation was also observed at 30 degrees C in the absence of AMP, particularly at higher enzyme concentration or in the presence of poly(ethylene glycol). Inactivation of the tetrameric enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme displayed Km values of 10 and 15 microM for fructose 6-phosphate and PPi, respectively, in the forward reaction, and of 35 and 590 microM for fructose 1,6-bisphosphate and Pi in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased Vmax. about 2-fold without changing the affinity for the substrates; its half-maximal effect was observed at 2 microM.

Topics: Adenosine Monophosphate; Animals; Chromatography, Gel; Chromatography, Ion Exchange; Detergents; Diphosphates; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fructosediphosphates; Kinetics; Macromolecular Substances; Molecular Weight; Naegleria fowleri; Octoxynol; Phosphofructokinase-1; Polyethylene Glycols; Spectrophotometry, Ultraviolet; Substrate Specificity; Thiocyanates

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was purified from the mung bean Phaseolus aureus. The enzyme is activated by fructose 2,6-bisphosphate at nanomolar concentrations. The enzyme exhibits Michaelis-Menten kinetics, and the reaction mechanism, deduced from initial velocity studies in the absence of inhibitors as well as product and dead-end inhibition studies, is rapid equilibrium random in the presence and absence of fructose 2,6-bisphosphate. In the direction of fructose 6-phosphate phosphorylation, saturating fructose 2,6-bisphosphate (1 microM) increases V congruent to 9-fold and increases V/KMgPPi and V/KF6P about 30-fold. In the reverse direction (phosphate phosphorylation), the same concentration of activator has little if any effect on V or the Km for inorganic phosphate (Pi) and Mg2+ but does increase V/KFBP about 42-fold. No changes were observed in any of the other rate constants. The binding affinity of fructose 2,6-bisphosphate to all enzyme forms is identical. The activator site of the mung bean PPi-PFK binds fructose 2,6-bisphosphate with a Kact of 30 nM with the 2,5-anhydro-D-glucitol 1,6-bisphosphate (the most effective analogue) 33-fold less tightly. Of the alkanediol bisphosphate series, 1,4-butanediol bisphosphate exhibited the tightest binding (Kact congruent to 3 microM). These and a series of other activating analogues are discussed in relation to the activator site.

Topics: Diphosphates; Enzyme Activation; Fabaceae; Fructosediphosphates; Hexosediphosphates; Kinetics; Mathematics; Phosphofructokinase-1; Plants, Medicinal; Structure-Activity Relationship; Sugar Phosphates

In the presence of pyrophosphate and uridine diphosphate, sucrose was cleaved to form glucose 1-phosphate and fructose with soluble extracts from sucrose importing plant tissues. The glucose 1-phosphate then was converted through glycolysis to triose phosphates in a pyrophosphate-dependent pathway which was activated by fructose 2,6-bisphosphate. Much less activity, less than 5%, was found in sucrose exporting tissue extracts from the same plants. These findings suggest that imported sucrose is metabolized in the cytoplasm of plant tissues by utilizing pyrophosphate and that sucrose metabolism is partially regulated by fructose 2,6-bisphosphate.

Topics: Biological Transport; Diphosphates; Fructosediphosphates; Glucosephosphates; Glycolysis; Hexosediphosphates; Plants; Sucrose; Trioses; Uridine Diphosphate