A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.     

Studies on the mechanism of Escherichia coli glucosamine-6-phosphate isomerase.

Escherichia coli glucosamine-6-phosphate isomerase is specific for removal of the 1-pro-R hydrogen of fructose 6-phosphate (fructose-6-P). The conversion of [2-3H]glucosamine-6-P to fructose-6-P plus ammonia is accompanied by 99% exchange of tritium with water and 0.6% transfer to C-1 of fructose-6-P. The enzyme is active toward alpha-glucosamine-6-P and apparently inactive toward the beta anomer. The combination of the above results supports a cisenolamine intermediate for the reaction. The labeling of substrate and product pools in tritiated water shows that the two halves of the reaction are each freely reversible. No single step appears to be rate determining. 2-Amino-2-deoxyglucitol-6-P is an unusually strong competitive inhibitor (K1 = 2 X 10(-7) M, compared with the Km = 4 X 10(-4) M for glucosamine-6-P), suggesting the enzyme has a strong affinity for the open-chain form of glucosamine-6-P.     

Resolution and characterization of multiple cytosolic phosphatases capable of hydrolyzing fructose-1,6-bisphosphate in spinach and soybean leaves

The apparent activity of cytoplasmic fructose bisphosphatase (EC 3.1.3.11) in crude extracts of spinach ( Spinacia oleracea L.) and soybean ( Glycine max [L.] Merr.) leaves was only partially dependent on Mg²⁺. At least two major non-chloroplastic fructose bisphosphatases that differed in dependence on Mg²⁺ were chromatographically resolved from spinach leaves. The Mg²⁺-dependent enzyme had an apparent Michaelis constant of 4 μM for fructose-1,6-P₂, was highly specific, and was strongly inhibited by fructose-2,6-P₂. Enzyme activity was inhibited by physiological levels of fructose-6-P.
Both species also contained at least one major enzyme, the activity of which was independent of Mg²⁺. These enzymes had pH optima near neutrality, Michaelis constants of 25 to 30 μM for fructose-1,6-P₂, and were inhibited by AMP. Although hexose monophosphates were not metabolized, the enzymes were not specific for fructose-1,6-P₂: phosphate was released from phosphoenolpyruvate and ribulose-1, 5-P₂, and with fructose-1,6-P₂, as substrate, Pi release was about 1.5-fold greater than fructose-6-P production. It is concluded that only the Mg²⁺-dependent fructose bisphosphatase, previously characterized, functions in the photosynthetic sucrose formation pathway. Inhibition of the Mg²⁺-dependent enzyme by fructose-6-P may be involved in regulation of sucrose formation.     

Inhibition of bovine hepatic fructose-1,6-diphosphatase by substrate analogs.

Purified bovine hepatic fructose-1,6-diphosphatase, which exhibits maximal activity at neutral pH, is competitively inhibited by several analogs of its substrate, fructose 1,6-diphosphate. These include glucose 1,6-diphosphate (Ki = 9.4 X 10(-5) M), hexitol 1,6-diphosphate (Ki = 2.3 X 10(-4) M), and 2,5-anhydro-D-mannitol 1,6-diphosphate (Ki = 3.3 X 10(-8) M), and 2,5-anhydro-D-glucitol 1,6-diphosphate (Ki = 5.5 X 10(-7) M). The Ki values for both 2,5-anhydro-D-mannitol 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate are lower than the Km of 1.4 X 10(-6) M for fructose 1,6-diphosphate. Since 2,5-anhydro-D-mannitol 1,6-diphosphate is an analog of the beta anomer of fructose 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate is an analog of the alpha anomer, the lower Ki for the mannitol analog may indicate that the beta anomer of fructose 1,6-diphosphate, which predominates in solution, is the true substrate. The substrate analog 1,5-pentanediol diphosphate inhibits slightly (K0.5 = 5 X 10(-3) M), but 1,4-cyclohexyldiol diphosphate does not. The Ki for product inhibition by sodium phosphate is 9.4 X 10(-3) M. 2,5-Anhydro-D-mannitol 1,6-diphosphate and alpha-D-glucose 1,6-diphosphate are substrates at pH 9.0, but not at pH 6.5.     

31P nuclear magnetic resonance spectroscopy studies of substrate and product binding to fructose-1,6-bisphosphatase.

The enzymatic hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P2) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (Pi), which is catalyzed by fructose-1,6-bisphosphatase, has been studied by 31P nuclear magnetic resonance spectroscopy (NMR). At pH 7.5 and 15 degrees C, the equilibrium constant for the central complex K'eq = [E.Fru-6-P.Pi]/[E.Fru-1,6-P2.H2O] is about 2. This observation is in harmony with results obtained with a number of Bi Bi enzyme systems for the determination of K'eq in which a variety of experimental techniques were used (Knowles, J.R. (1980) Annu. Rev. Biochem. 49, 877-919). Significant changes in 31P NMR chemical shifts were observed for both the substrate, Fru-1,6-P2, and the product, Fru-6-P, when bound to the enzyme relative to ligand free in solution. The chemical shifts of the substrate and product were altered further in the presence of Mg2+, the catalytic divalent metal ion. The chemical shifts caused by the addition of metal ion can be reversed in the presence of trans-1,2-diaminocyclohexane- N,N,N',N'-tetraacetic acid (CDTA) or AMP. In the presence of the metal ion chelator or the nucleotide, the substrate had a chemical shift that was about the same as that observed in the absence of metal ion. On the basis of these observations we suggest that AMP and CDTA exhibit similar effects, i.e. they both remove the catalytic metal ion from the enzyme. This finding is supportive of the suggestion (Scheffler, J. E., and Fromm, H.J. (1986) Biochemistry 25, 6659-6665; Liu, F., and Fromm, H.J. (1990) J. Biol. Chem. 265, 7401-7406) that the role of AMP in the regulation of fructose-1,6-bisphosphatase is to prevent binding of the divalent metal activator to the enzyme.     

The occurrence of inorganic pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase in higher plants. I. Initial characterization of partially purified enzyme from Sanseviera trifasciata leaves

Among 30 plant species examined, the PPi-phosphofructokinase (EC 2.7.1.90) was found in leaves of 21 plants. Some of the plants exhibit no activity of ATP-dependent phosphofructokinase but display only activity of PPi-phosphofructokinase. A partly purified preparation of PPi-phosphofructokinase with specific activity of 8.4 Hmol (mg protein)⁻¹ min⁻¹ was obtained from Sanseviera trifasciata leaves. The enzyme is restricted to the cytoplasm, it exhibits pronounced substrate specifity, requires Mg²⁺ ions, is inhibited by AMP, PEP, methylenediphosphonate and stabilized by mercaptoethanol. At pH 7.8 with 1.5 m M MgCl₂ the following K_M values were observed: pyrophosphate, 0.58 m M ; fructose 6-phosphate, 0.8 m M . The K_M values for substrates of reverse reaction (pH 7.3; 2 m M MgCl₂) are of the same order of magnitude: 0.83 m M for fructose 1,6-diphosphate, and 0.14 m M for orthophosphate. The molecular weight of the studied enzyme is about 125 000 dalton as estimated by gel filtration.     

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.     

Methylglyoxal is an intermediate in the biosynthesis of 6-deoxy-5-ketofructose-1-phosphate: a precursor for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii

A biosynthetic pathway is proposed for creating 6-deoxy-5-ketofructose-1-phosphate (DKFP), a precursor sugar for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii. First, two possible routes were investigated to determine if a modified, established biosynthetic pathway could be responsible for generating 6-deoxyhexoses in M. jannaschii. Both the nucleoside diphosphate mannose pathway and a pathway involving nucleoside diphosphate derivatives of fructose-1-P, fructose-2-P, or fructose-1,6-bisP were tested and eliminated. The established pathways did not produce the expected intermediates nor did the anticipated enzymes have the predicted enzymatic activities. Because neither anticipated pathway could produce DKFP, M. jannaschii glucose-6-P metabolism was studied in detail to establish exactly how glucose-6-P is converted into DKFP. This detailed analysis showed that methylglyoxal and a fructose-1-P- or fructose-1,6-bisP-derived dihydroxyacetone-P fragment are key intermediates in DKFP production. Glucose-6-P readily converts to fructose-6-P, which in turn converts to fructose-1,6-bisP. Fructose-6-P and fructose-1,6-bisP convert into glyceraldehyde-3-P (Ga-P-3), which converts into methylglyoxal by a 2,3-elimination of phosphate. The MJ1585-derived enzyme catalyzes the condensation of methylglyoxal with a dihydroxyacetone-P fragment, which is derived from fructose-1-P and/or fructose-1,6-bisP, generating DKFP. The elimination of phosphate from Ga-P-3 proceeds by both enzymatic and chemical routes in cell extracts, producing sufficient concentrations of methylglyoxal to support the reaction. This work is the first report of methylglyoxal functioning in central metabolism.     

Substrate specificity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase from potato tuber

The aim of this work was to establish the precise ionic form of the reactants used by pyrophosphate:fructose-6-phosphate phosphotransferase. The enzyme was purified to near-homogeneity from potato (Solanum tuberosum L.) tubers. Changes in enzyme activity when the pH of the assay and the concentration of fructose 6-phosphate, pyrophosphate, and magnesium are varied independently indicate that fructose 6-phosphate²⁻ and MgP₂O₇²⁻ are the reacting species in the glycolytic direction. Analogous experiments with fructose 1,6-bisphosphate, inorganic phosphate, and magnesium demonstrate that the enzyme uses fructose 1,6-bisphosphate⁴⁻, HPO₄²⁻, and Mg²⁺ in the gluconeogenic direction. The ionic species used in the glycolytic direction are comparable with those required by bacterial ATP-dependent phosphofructokinase. This is consistent with the proposal that the active site of pyrophosphate:fructose-6-phosphate phosphotransferase in plants is equivalent to that of the bacterial phosphofructokinase (SM Carlisle et al. [1990] J Biol Chem 265: 18366-18371).     

Activation of muscle phosphofructokinase by fructose 2,6-bisphosphate and fructose 1,6-bisphosphate is differently affected by other regulatory metabolites

Fructose-2,6-P2 and fructose-1,6-P2 are strong activators of muscle phosphofructokinase. They have been shown to be competitive in binding studies, and it is generally thought that they affect the physical and catalytic properties of the enzyme in the same manner. However, there are indications in published data that the effects of the two fructose bisphosphates on phosphofructokinase are not identical. To examine this possibility, the kinetics of activation of rat skeletal muscle phosphofructokinase by the two fructose bisphosphates were compared in the presence of other regulatory metabolites. Citrate greatly increased the K0.5 of the enzyme for fructose-2,6-P2, with little effect on the maximum activation. In contrast, citrate greatly decreased the maximum activation by fructose-1,6-P2, with only a small effect on the K0.5. Changes in the concentrations of the inhibitor ATP or the activator AMP similarly altered the K0.5 for fructose-2,6-P2, but altered the maximum activation by fructose-1,6-P2. Finally, when fructose-1,6-P2 was added in the presence of a given concentration of fructose-2,6-P2, phosphofructokinase activity was decreased if the activation by fructose-2,6-P2 alone was greater than the maximum activation by fructose-1,6-P2 alone. These results are consistent with competition of the two fructose bisphosphates for the same binding site, but indicate that the conformational changes produced by their binding are different.     

Mechanism of action of 2,5-anhydro-D-mannitol in hepatocytes. Effects of phosphorylated metabolites on enzymes of carbohydrate metabolism

Isolated rat hepatocytes convert 2,5-anhydromannitol to 2,5-anhydromannitol-1-P and 2,5-anhydromannitol-1,6-P2. Cellular concentrations of the monophosphate and bisphosphate are proportional to the concentration of 2,5-anhydromannitol and are decreased by gluconeogenic substrates but not by glucose. Rat liver phosphofructokinase-1 phosphorylates 2,5-anhydromannitol-1-P; the rate is less than that for fructose-6-P but is stimulated by fructose-2,6-P2. At 1 mM fructose-6-P, bisphosphate compounds activate rat liver phosphofructokinase-1 in the following order of effectiveness: fructose-2,6-P2 much greater than 2,5-anhydromannitol-1,6-P2 greater than fructose-1,6-P2 greater than 2,5-anhydroglucitol-1,6-P2. High concentrations of fructose-1,6-P2 or 2,5-anhydromannitol-1,6-P2 inhibit phosphofructokinase-1. Rat liver fructose 1,6-bisphosphatase is inhibited competitively by 2,5-anhydromannitol-1,6-P2 and noncompetitively by 2,5-anhydroglucitol-1,6-P2. The AMP inhibition of fructose 1,6-bisphosphatase is potentiated by 2,5-anhydroglucitol-1,6-P2 but not by 2,5-anhydromannitol-1,6-P2. Rat liver pyruvate kinase is stimulated by micromolar concentrations of 2,5-anhydromannitol-1,6-P2; the maximal activation is the same as for fructose-1,6-P2. 2,5-Anhydroglucitol-1,6-P2 is a weak activator. 2,5-Anhydromannitol-1-P stimulates pyruvate kinase more effectively than fructose-1-P. Effects of glucagon on pyruvate kinase are not altered by prior treatment of hepatocytes with 2,5-anhydromannitol. Pyruvate kinase from glucagon-treated hepatocytes has the same activity as the control pyruvate kinase at saturating concentrations of 2,5-anhydromannitol-1,6-P2 but has a decreased affinity for 2,5-anhydromannitol-1,6-P2 and is not stimulated by 2,5-anhydromannitol-1-P. The inhibition of gluconeogenesis and enhancement of glycolysis from gluconeogenic precursors in hepatocytes treated with 2,5-anhydromannitol can be explained by an inhibition of fructose 1,6-bisphosphatase, an activation of pyruvate kinase, and an abolition of the influence of phosphorylation on pyruvate kinase.     

Phosphorylation and inactivation of yeast fructose-1,6-bisphosphatase by cyclic AMP-dependent protein kinase from yeast

Purified fructose-1,6-bisphosphatase from Saccharomyces cerevisiae was phosphorylated in vitro by purified yeast cAMP-dependent protein kinase. Maximal phosphorylation was accompanied by an inactivation of the enzyme by about 60%. In vitro phosphorylation caused changes in the kinetic properties of fructose-1,6-bisphosphatase: 1) the ratio R(Mg2+/Mn2+) of the enzyme activities measured at 10 mM Mg2+ and 2 mM Mn2+, respectively, decreased from 2.6 to 1.2; 2) the ratio R(pH 7/9) of the activities measured at pH 7.0 and pH 9.0, respectively, decreased from 0.62 to 0.38, indicating a shift of the pH optimum to the alkaline range. However, the affinity of the enzyme for its inhibitors fructose-2,6-bisphosphate (Fru-2,6-P2) and AMP, expressed as the concentration required for 50% inhibition, was not changed. The maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase was 0.6-0.75 mol/mol of the 40-kDa subunit. Serine was identified as the phosphate-labeled amino acid. The initial rate of in vitro phosphorylation of fructose-1,6-bisphosphatase, obtained with a maximally cAMP-activated protein kinase, increased when Fru-2,6-P2 and AMP, both potent inhibitors of the enzyme, were added. As Fru-2,6-P2 and AMP did not affect the phosphorylation of histone by cAMP-dependent protein kinase, the inhibitors must bind to fructose-1,6-bisphosphatase in such a way that the enzyme becomes a better substrate for phosphorylation. Nevertheless, Fru-2,6-P2 and AMP did not increase the maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase beyond that observed in the presence of cAMP alone.     

Modulation of muscle phosphofructokinase at physiological concentration of enzyme

Two approaches have been used to study the allosteric modulation of phosphofructokinase at physiological concentration of enzyme; a "slow motion" approach based on the use of a very low Mg2+/ATP ratio to conveniently lower Vmax, and the addition of polyethylene glycol as a "crowding" agent to favor aggregation of diluted enzyme. At 0.6 mg/ml muscle phosphofructokinase exhibited a drastic decrease in the ATP inhibition and the concomitant increase in the apparent affinity for fructose-6-P, as compared to a 100-fold diluted enzyme. Similar results were obtained with diluted enzyme in the presence of 10% polyethylene glycol (Mr = 6000). Results with these two approaches in vitro were essentially similar to those previously observed in situ (Aragón, J. J., Felíu, F. E., Frenkel, R., and Sols, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6324-6328), indicating that the enzyme is strongly dependent on homologous interactions at physiological concentrations. With polyethylene glycol it was observed that within the physiological range of concentration of substrates and the other positive effectors, fructose-2,6-P2 still activates the liver phosphofructokinase although it no longer significantly affects the muscle isozyme. In the presence of polyethylene glycol, muscle phosphofructokinase can approach its maximal rate even in the presence of physiologically high concentrations of ATP. Three minor activities of muscle phosphofructokinase have been studied at high enzyme concentration: the hydrolysis of MgATP (ATPase) and fructose-1,6-P2 (FBPase), produced in the absence of the other substrate, and the reverse reaction from MgADP and fructose-1,6-P2. The kinetic study of these activities has allowed a new insight into the mechanisms involved in the modulation of phosphofructokinase activity. The binding of (Mg)ATP at its regulatory site reduces the ability of the enzyme to cleave the bond of the terminal phosphate of MgATP at the substrate site. The positive effectors (Pi, cAMP, NH+4, fructose-1,6-P2, and fructose-2,6-P2) decrease the inhibitory effect of MgATP. Citrate and fructose-2,6-P2 both act as mechanistically "secondary" effectors in the sense that citrate does not inhibit and fructose-2,6-P2 does not activate the FBPase activity, requiring both the presence of ATP to affect the enzyme activity. In conclusion it appears that the regulatory behavior of mammalian phosphofructokinases is utterly dependent on the fact of their high concentrations in vivo.     

Selective thiol group modification renders fructose-1,6-bisphosphatase insensitive to fructose 2,6-bisphosphate inhibition

Limited treatment of native pig kidney fructose-1,6-bisphosphatase (50 microM enzyme subunit) with [14C]N-ethylmaleimide (100 microM) at 30 degrees C, pH 7.5, in the presence of AMP (200 microM) results in the modification of 1 reactive cysteine residue/enzyme subunit. The N-ethylmaleimide-modified fructose-1,6-bisphosphatase has a functional catalytic site but is no longer inhibited by fructose 2,6-bisphosphate. The enzyme derivative also exhibits decreased affinity toward Mg2+. The presence of fructose 2,6-bisphosphate during the modification protects the enzyme against the loss of fructose 2,6-bisphosphate inhibition. Moreover, the modified enzyme is inhibited by monovalent cations, as previously reported (Reyes, A., Hubert, E., and Slebe, J.C. (1985) Biochem. Biophys. Res. Commun. 127, 373-379), and does not show inhibition by high substrate concentrations. A comparison of the kinetic properties of native and N-ethylmaleimide-modified fructose-1,6-bisphosphatase reveals differences in some properties but none is so striking as the complete loss of fructose 2,6-bisphosphate sensitivity. The results demonstrate that fructose 2,6-bisphosphate interacts with a specific allosteric site on fructose-1,6-bisphosphatase, and they also indicate that high levels of fructose 1,6-bisphosphate inhibit the enzyme by binding to this fructose 2,6-bisphosphate allosteric site.     

Selective modification of rabbit liver fructose bisphosphatase

Chemical modification of rabbit liver fructose 1,6-bisphosphatase by 5,5′-dithiobis-(2-nitrobenzoic acid) results in thiolation of four highly reactive sulfhydryl groups and a diminished sensitivity to AMP inhibition but not loss of enzyme activity. Ethoxyformylation of the histidine groups of fructose 1,6-bisphosphatase does not result in a sharp loss of activity until at least 4 or 5 of the 13 residues have reacted. Exhaustive formylation does abolish the enzyme's activity. These four most reactive sulfhydryl groups and the one or two least easily modified histidine moieties (those responsible for activity) can be protected against modification by fructose-1,6-P₂ and to a lesser extent by fructose-6-P. The binding of fructose-1,6-P₂ to fructose 1,6-bisphosphatase, however, depends on the presence of structural metal ion since EDTA which removes all endogenous Zn²⁺ from the protein prevents binding of fructose-1, 6-P₂ to the enzyme.     

Reaction mechanism of erythrocyte phosphofructokinase.

The reaction mechanism of erythrocyte phosphofructokinase (PFK) was investigated by the initial velocity and the product inhibition. Intersecting lines obtained with initial velocity studies are consistent with a sequential mechanism and the formation of ternary complex as an intermediate. The product inhibition studies support an ordered Bi Bi mechanism in which fructose 6 phosphate (F6P) is the first substrate binding and adenosine diphosphate (ADP) is dissociated from the enzyme before fructose-1,6-P2 (FDP).     

Biosynthesis of bacterial glycogen: characterization of adenosine diphosphate glucose synthetases from Enterobacter hafniae and Aeromonas hydrophila

Enterobacter hafniae and Aeromonas hydrophila ADPglucose synthetases were purified approximately 39-and 61-fold, respectively, over the crude extract. Both enzymes were heat stable at 60°C in the presence of inorganic phosphate. The molecular weights of both enzymes were approximately 200,000 which are similar to other enteric ADPglucose synthetases studied. Based on kinetic results obtained from the partially purified enzymes, the E. hafniae enzyme is activated twofold by phospho-enolpyruvate while the A. hydrophila enzyme is activated twofold by fructose 6-P and 1.5-fold by fructose 1,6 bis-phosphate. The E. hafniae enzyme activity is strongly inhibited by AMP and ADP and the inhibition can be partially reversed by P-enolpyruvate. ADP is the most effective inhibitor of the A. hydrophila enzyme and its inhibiton can be partially overcome by the presence of the activators fructose 6-P and fructose 1,6-P₂. These kinetic results show that the allosteric properties of the E. hafniae enzyme are distinctly different from the ADPglucose synthetases of those previously studied from bacteria of the genus Enterobacter. Although the A. hydrophila enzyme is activated by fructose 1,6-P₂, its allosteric properties are quite different than those observed for ADPglucose synthetase of the Enterobacteriaceae.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - glucose 1-P glucose 1-phosphate - Bicine N,N-bis(2 hydroxyethyl)glycine - fructose 6-P fructose 6-phosphate - Mes 2(N-morpholino)-ethane sulfonic acid - fructose 1,6-P₂ fructose 1,6 bis-phosphate - DTE dithioerythritol; pyridoxal-P, pyridoxal-phosphate - fructose 1-P fructose 1-phosphate - P-enolpyruvate phospho-enolpyruvate - 1,6 hexanediol bis-P 1,6 hexanediol bis-phosphate; glucose 6-P, glucose 6-phosphate - dihydroxyacetone-P dihydroxyacetone phosphate - 1-glycerol-3-P 1-glycerol-3-phosphate - erythrose 4-P erythrose 4-phosphate - 2-P-glycerate 2-phosphoglycerate - sedoheptulose 1,7-P₂ sedoheptulose 1,7 bis-phosphate - 3-P-glycerate 3-phosphoglycerate - mannose-6-P mannose-6-phosphate     

Acute effects of oral and intravenous ethanol on rat hepatic enzyme activities.

1. Oral administration of ethanol (3 ml) of 95% in 12 ml total volume over a two day period) significantly decrease plasma glucose and insulin levels and the activities of two key gluconeogenic enzymes, pyruvate carboxylase (pyruvate: CO2 ligase (ADP), EC 6.4.1.1) and fructose diphosphatase, (D-Fru-1,6-P2 1-phosphohydrolase, EC 3.1.3.11), and one glycolytic enzyme, fructose-1,6-P2 aldolase (Fru-1,6-P2 D-glyceraldehyde-3-P lyase, EC 4.1.2.13). In each instance, the administration of 2400 mug daily of oral folate in conjuction with the ethanol prevented these alterations in carbohydrate metabolism. 2. Intravenous injection of ethanol produced a rapid decrease (within 10--15 min) in the activities of hepatic phosphofructokinase, (ATP:D-fructose-6-phosphate 6-phosphotransferase, EC 2.7.1.11), pyruvate kinase, (ATP:pyruvate phosphotransferase, EC 2.7.1.40), fructose diphosphatase and fructose-1,6-P2 aldolase. 3. Intravenous ethanol significantly increased hepatic cyclic AMP concentration approximately 60% within 10 min, while oral ethanol did not alter hepatic cyclic AMP concentrations. 4. These data confirm the known antagonism ethanol and folate and suggest that oral folate might offer a protective effect against hypoglycemia in rats receiving ethanol.     

Potentiation by glucose metabolites of inositol trisphosphate-induced calcium mobilization in permeabilized rat pancreatic islets

Saponin-permeabilized rat pancreatic islets degraded exogenously added inositol 1,4,5-trisphosphate (IP3), and degradation was inhibited in the presence of either fructose 1,6-bisphosphate or diphosphoglycerate. The addition of either fructose-1,6-P2 or diphosphoglycerate to 45Ca2+-labeled permeabilized islets potentiated 45Ca2+ release caused by IP3 (by either exogenously added IP3 or IP3 generated endogenously in the presence of carbachol or guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). The effect of diphosphoglycerate and fructose-1,6-P2 on 45Ca2+ release correlated well with the effects of these agents on the recovery of radioactivity in IP3. These results further support our previous proposal that in pancreatic islets intracellular calcium mobilization may be sustained in part via the inhibition of IP3 degradation by metabolites produced during stimulation with insulinotropic concentrations of glucose (Rana, R.S., Sekar, M.C., Hokin, L.E., and MacDonald, M.J. (1986) J. Biol. Chem. 261, 5237-5240).     

PPi-dependent phosphofructotransferase (phosphofructokinase) activity in the mollicutes (mycoplasma) Acholeplasma laidlawii.

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.