Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate in equilibrium fructose 6-phosphate, [32P]Pi in equilibrium MgPPi, and Mg[32P]PPi in equilibrium fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate-product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi, in agreement with initial velocity studies [Bertagnolli, B.L., & Cook, P.F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pi nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.     

A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate

Pyrophosphate : fructose-6-phosphate phosphotransferase (PPi-PFK) has been purified 150-fold from potato tubers and the kinetic properties of the purified enzyme have been investigated both in the forward and the reverse direction. Saturation curves for fructose 6-phosphate and also for fructose 1,6-bisphosphate were sigmoidal whereas those for PPi and Pi were hyperbolic. In the presence of fructose 2,6-bisphosphate, the affinity for fructose 6-phosphate and for fructose 1,6-bisphosphate were greatly increased and the kinetics became Micha?lian. The effect of fructose 2,6-bisphosphate was increased by the presence of fructose 6-phosphate and decreased by the presence of Pi. Consequently, the Ka for fructose 2,6-bisphosphate was as low as 5 nM for the forward reaction and reached 150 nM for the reverse reaction. On the basis of these properties, a procedure allowing one to measure fructose 2,6-bisphosphate in amounts lower than a picomole, is described.     

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.     

Kinetic studies on the activation of pyrophosphate-dependent phosphofructokinase from mung bean by fructose 2,6-bisphosphate and related compounds

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.     

Properties of Pyrophosphate:Fructose-6-Phosphate Phosphotransferase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90) from endosperm of developing wheat (Triticum aestivum L.) grains was purified to apparent homogeneity with about 52% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and gel filtration through Sepharose-CL-6B. The purified enzyme, having a molecular weight of about 170,000, was a dimer with subunit molecular weights of 90,000 and 80,000, respectively. The enzyme exhibited maximum activity at pH 7.5 and was highly specific for pyrophosphate (PPi). None of the nucleoside mono-, di- or triphosphate could replace PPi as a source of energy and inorganic phosphate (Pi). Similarly, the enzyme was highly specific for fructose-6-phosphate. It had a requirement for Mg²⁺ and exhibited hyperbolic kinetics with all substrates including Mg²⁺. K_m values as determined by Lineweaver-Burk plots were 322, 31, 139, and 129 micromolar, respectively, for fructose-6-phosphate, PPi, fructose-1,6-bisphosphate and Pi. 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 its substrates. Initial velocity studies indicated kinetic mechanism to be sequential. At saturating concentrations of fructose-2,6-bisphosphate (1 micromolar), Pi strongly inhibited PFP; the inhibition being mixed with respect to both fructose-6-phosphate and PPi, with K_i values of 0.78 and 1.2 millimolar, respectively. The inhibition pattern further confirmed the mechanism to be sequential with random binding of the substrates. Probable role of PFP in endosperm of developing wheat grains (sink tissues) is discussed.     

Model for prebiotic pyrophosphate formation: Condensation of precipitated orthophosphate at low temperature in the absence of condensing or phosphorylating agents

Summary The formation of pyrophosphate (PPi) by condensation of orthophosphate (Pi) at low temperature (37°C) in the absence of condensing or phosphorylating agents could have been an ancient process in chemical evolution. In the present investigation the synthesis of³²PPi from³²Pi was carried out at pH 8.0 and PPi was found in larger amounts in the presence of insoluble Pi (with calcium or manganese ions) than in its absence (with magnesium ions, or with no divalent cations present). After 10 days of incubation in the presence of precipitated calcium phosphate, about 1.6 nmol/ml of PPi was formed (0.057% yield relative to insoluble Pi). The hypothesis that the reaction is dependent on precipitated Pi was reinforced by the effect of adding dimethyl sulfoxide (2.1–9.9 M) in the presence of magnesium ions: the amount of magnesium phosphate precipitated in the presence of the organic solvent was proportional to the amount of PPi formed. The large and negative activation entropies found in aqueous media with calcium ions and in a medium containing 11.3 M dimethyl sulfoxide with magnesium ions suggest that the reaction was favored by a hydrophobic phenomenon at the surface of solid Pi. This reaction could serve as a model for prebiotic formation of PPi.     

菠萝叶片依赖焦磷酸的磷酸果糖激酶的纯化及其特性

经硫酸铵分部,DEAE—纤维素、羟基磷灰石、Sephadex G—200及磷酸纤维素柱层析,从菠萝叶片分离得到电泳均一的依赖焦磷酸的磷酸果糖激酶(PFP)。SDS电泳图谱表明有一条分子量为62kD的主带和一条57 kD的弱带。Fru—2,6—P_2对酶的正反应活性有促进作用。动力学研究表明,Fru—2,6—P_2增加V_(max)及酶对底物Fru—6—P和Mg~(2+)的亲和性。     

Pyrophosphate synthesis from phospho(enol)pyruvate catalyzed by precipitated magnesium phosphate with ”enzyme-like” activity

Summary The enzyme-like kinetic properties of precipitated magnesium phosphate as a catalyst for formation of pyrophosphate (PPi) from phospho (enol)pyruvate (PEP) are described. This synthesis occurs at a low temperature (37°C) and represents a model that may help us understand the relevance to chemical evolution of minerals as ancient catalysts whose functions could have been taken over by contemporary enzymes. An insoluble Pi.Mg matrix was formed in a medium with 80% of the water replaced by dimethyl sulfoxide as a way of simulating conditions in a drying pond. Phospho(enol)pyruvate adsorbs onto the Pi.Mg surface according to a Langmuir isotherm, and the PEP concentration dependence of PPi formation follows a Michaelian-like function. A yield of 33% for transformation of the initially adsorbed PEP into PPi was attained after 4 days of incubation with equimolecular concentrations of Pi, MgCl₂, and PEP. The magnesium concentration dependence for Pi and Mg precipitation, for adsorption of PEP onto solid Pi.Mg, and for PPi formation showed complex cooperative behavior. These results taken as a whole lead to the conclusion that the Pi.Mg surface not only provides a reactant for PPi formation but also catalyzes the reaction.Offprint requests to: A. Vieyra     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

Properties and reaction mechanism of C4 leaf pyruvate,Pi dikinase

The properties and reaction mechanism of maize leaf pyruvate,Pi dikinase are described. Km values were determined for the forward reaction substrates, pyruvate, ATP, and Pi, at pH 7.4 and 8.0 and for reverse reaction substrates at pH 7.4. Enzyme activity was almost totally dependent on added monovalent cations in both directions. NH+4 was most effective, with Ka values of about 0.38 mM for the forward reaction and 2 mM for the reverse reaction. K+ also completely activated the enzyme in the forward direction (Ka = 8 mM) but only partially activated in the reverse direction. Na+ had little effect on either reaction. The pH optimum for the forward reaction was about 8.2; the reverse reaction optimum was about 6.9. Maximum activity for the reverse direction was about twice the maximum forward direction rate. From data on the requirements for the ATP-AMP exchange reaction, on the mechanism of inhibition of the forward reaction by PEP, AMP, and PPi, and from the kinetics of the interaction of varying certain substrate pairs, it was concluded that the maize leaf pyruvate,Pi dikinase reaction proceeded by the two-step Bi Bi Uni Uni mechanism. This differs from the mechanism of catalysis by the bacterial enzyme.     

Theoretical analysis of distribution of [18O]Pi species during exchange with water. Application to exchanges catalyzed by yeast inorganic pyrophosphatase

A theoretical analysis has been derived which allows the analytical calculation of the complete distribution of 18O-labeled Pi species expected to occur during medium Pi equilibrium HOH exchange of [18O]Pi and to be produced by intermediate Pi equilibrium HOH exchange during net hydrolysis of [18O]PPi or other labeled phosphate compounds. The observed distributions with catalysis by yeast inorganic pyrophosphatase are found to agree closely with the theoretical values indicating that the exchange reaction can be adequately described by a unique value of the partitioning of bound Pi between release from the enzyme versus formation of bound PPi with loss of an oxygen to the water. The limitations on the exclusion of other mechanisms are discussed. The extent of this partitioning does change, however, under some experimental conditions. At low pH, with activation by Mg2+ or Mn2+, the relative rate of release of Pi is found to increase. The extent of exchange is also dependent on the nature of the activating metal, being greatest with Co2+. During PPi hydrolysis with PPi in excess over Mg2+, a shift to lower extents of exchange is observed.     

Characterization of phosphate:hexose 6-phosphate antiport in membrane vesicles of Streptococcus lactis

Membrane vesicles of Streptococcus lactis were used to characterize a novel anion exchange involving phosphate and sugar 6-phosphates. For vesicles loaded with 50 mM phosphate at pH 7, homologous phosphate:phosphate exchange had a maximal rate of 130 nmol/min/mg of protein and a Kt of 0.21 mM external phosphate; among phosphate analogues tested, only arsenate replaced phosphate. Heterologous exchange was studied by 2-deoxyglucose 6-phosphate entry into phosphate-loaded vesicles; this reaction had a maximal velocity of 31 nmol/min/mg of protein and a Kt of 26 microM external substrate. Sugar phosphate moved intact during this exchange, since its entry led to loss of internal 32Pi without transfer of 32P to sugar phosphate. Inhibitions of phosphate exchange suggested that the preferred sugar phosphate substrates were (Kiapp): glucose, 2-deoxyglucose, and mannose 6-phosphates (approximately 20 microM) greater than fructose 6-phosphate (150 microM) greater than glucosamine 6-phosphate (420 microM) greater than alpha-methylglucoside 6-phosphate (740 microM). Stoichiometry for phosphate:2-deoxyglucose 6-phosphate antiport was 2:1 at pH 7, and since initial rates of exchange were unaffected by charge carrying ionophores (gramicidin, valinomycin, a protonophore), this unequal stoichiometry indicated the electroneutral exchange of two monovalent phosphates for a single divalent sugar phosphate.     

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.     

Enzymatic Studies on the Conversion of Erythritol Fermentation to d-Mannitol Fermentation in n-Alkane-grown Yeast Candida zeylanoides

In the polyol fermentation by Candida zeylanoides KY6166, which occurred preferentially by keeping the pH of medium at acidic side (below 4.0), phosphate ion played a precise role in the conversion of erythritol fermentation to d-mannitol fermentation. Enzymatic studies on the conversion mechanism provided the following evidences.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphate ion.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphateion. From these results, the alteration from erythritol fermentation to mannitol fermentation by phosphate ion was explained as the result of the change in the level of erythrose 4-phosphate and fructose 6-phosphate which was caused by the inhibition of transketolase.     

Role of mono- and divalent metal cations in the catalysis by yeast aldolase

The rate of deuterium exchange between [1-(S)-2H]dihydroxyacetone 3-phosphate and the solvent catalyzed by native and metal-substituted yeast aldolases has been measured. In the presence of 0.1 M potassium acetate at 15 degrees C, pH 7.3, the deuterium exchange reaction catalyzed by native yeast aldolase has a kcat of 95 s-1. In contrast to the 7-fold activity enhancement by 0.1 M potassium ion (relative to 0.1 M sodium ion) of the cleavage of D-fructose 1,6-bisphosphate catalyzed by native yeast aldolase, a negligible (1.1-fold) activation by 0.1 M potassium ion is observed in the rate of dedeuteration of [1(S)-2H]dihydroxyacetone 3-phosphate. The order of reactivity of the yeast metalloaldolases in the deuterium exchange roughly parallels that seen in the fructose bisphosphate cleavage reaction. These findings suggest that the carbonyl groups of enzyme-bound D-fructose 1,6-bisphosphate and dihydroxyacetone phosphate are both polarized by the active site divalent metal cation. A mechanistic formulation consistent with the results of this and the previous paper is presented.     

Steady state and exchange kinetics of pyruvate, phosphate dikinase from Propionibacterium shermanii.

Evidence is presented based on requirements for exchange in the partial reactions, initial velocity and exchange kinetics and product inhibition, that the pyruvate, phosphate dikinase reaction of propionibacteria occurs by a nonclassical Tri Uni Uni Ping Pong mechanism. The mechanism involves a pyrophosphoryl enzyme, a phosphoryl enzyme, and the free enzyme, and three functionally distinct and independent substrate sites. On the first site, there is pyrophosphorylation of the enzyme by ATP with subsequent release of AMP. The pyrophosphoryl moiety then reacts at the second site with Pi yielding the product PPi and the phosphoryl from of the enzyme. At the third site pyruvate is phosphorylated yielding P-enolpyruvate and the free enzyme. The three catalytic sites are proposed to be linked by a histidyl residue which functions as a pyrophosphoyrl- and phosphoryl-carrier between the three sites. This proposal is based on the following observations. (A) The patterns of the double reciprocal plots of the initial velocities were all parallel; (b) product inhibition between each pair of substrates and products of the three partial reactions were competitive, i.e. ATP against AMP, Pi against PPi, and pyruvate against P-enolpyruvate; (c) the other product inhibitions, with one exception, were noncompetitive as required by the nonclassical ping-pong mechanism; (d) ATP or P-enolpyruvate was required for the Pi in equilibrium PPi exchange reaction which is in accord with the participation of a pyrosphosphoryl or phosphoryl form of the enzyme in this exchange; (e) the ATP in equilibrium AMP exchange and pyruvate in equilibrium P-enolpyruvate exchange did not require additional substrates. In addition, the inhibition and participation in the exchange reactions of the alpha,beta and beta,gamma-methylene analogues of ATP and of the methylene analogue of inorganic pyrophosphate were investigated and the results were in accord with the proposed mechanism. The combined evidence provides a well documented example of a three site nonclassical Tri Uni Uni Ping Pong mechanism.     

Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichia coli and is related to a novel group of bacterial transaldolases

We have cloned an open reading frame from the Escherichia coli K-12 chromosome that had been assumed earlier to be a transaldolase or a transaldolase-related protein, termed MipB. Here we show that instead a novel enzyme activity, fructose-6-phosphate aldolase, is encoded by this open reading frame, which is the first report of an enzyme that catalyzes an aldol cleavage of fructose 6-phosphate from any organism. We propose the name FSA (for fructose-six phosphate aldolase; gene name fsa). The recombinant protein was purified to apparent homogeneity by anion exchange and gel permeation chromatography with a yield of 40 mg of protein from 1 liter of culture. By using electrospray tandem mass spectroscopy, a molecular weight of 22,998 per subunit was determined. From gel filtration a size of 257,000 (+/- 20,000) was calculated. The enzyme most likely forms either a decamer or dodecamer of identical subunits. The purified enzyme displayed a V(max) of 7 units mg(-)1 of protein for fructose 6-phosphate cleavage (at 30 degrees C, pH 8.5 in 50 mm glycylglycine buffer). For the aldolization reaction a V(max) of 45 units mg(-)1 of protein was found; K(m) values for the substrates were 9 mm for fructose 6-phosphate, 35 mm for dihydroxyacetone, and 0.8 mm for glyceraldehyde 3-phosphate. FSA did not utilize fructose, fructose 1-phosphate, fructose 1,6-bisphosphate, or dihydroxyacetone phosphate. FSA is not inhibited by EDTA which points to a metal-independent mode of action. The lysine 85 residue is essential for its action as its exchange to arginine (K85R) resulted in complete loss of activity in line with the assumption that the reaction mechanism involves a Schiff base formation through this lysine residue (class I aldolase). Another fsa-related gene, talC of Escherichia coli, was shown to also encode fructose-6-phosphate aldolase activity and not a transaldolase as proposed earlier.     

Proton transport in maize tonoplasts supported by fructose-1,6-bisphosphate cleavage. Pyrophosphate-dependent phosphofructokinase as a pyrophosphate-regenerating system

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.     

Characterization of cardiac sarcoplasmic reticulum ATP-ADP phosphate exchange and phosphorylation of the calcium transport adenosine triphosphatase.

1. The terminal phosphate of (gamma-32P)ATP is rapidly incorporated into cardiac sarcoplasmic reticulum membranes (0.7--1.3 mumol/g protein) in the presence of calcium and magnesium. Cardiac sarcoplasmic reticulum membranes catalize an ATP-ADP phosphate exchange in the presence of calcium and magnesium. 2. Half-maximum activation of the phosphoprotein formation and ATP-ADP phosphate exchange is reached at an ionized calcium concentration of about 0.3 muM. The Hill coefficients are 1.3. 3. Transphosphorylation and ATP-ADP phosphate exchange require magnesium and are maximally activated at magnesium concentrations close to or equal to the ATP concentration. 4. The phosphoprotein level is reduced to about 45% at an ADP/ATP ratio of 0.1. The rate of calcium-dependent ATP splitting declines, whilst the rate of the calcium-dependent ATP-ADP phosphate exchange increases when the ADP/ATP ratio is varied from 0.1 to 1. The sum of both, the rate of ATP splitting and the rate of ADP-ATP phosphate exchange remains constant. 5. Phosphoprotein formation and ATP-ADP phosphate exchange are not affected by azide, dinitrophenol, dicyclohexyl carbodiimide and oubain, whilst both activities are reduced by blockade of -SH groups localized on the outside of the sarcoplasmic reticulum membrane. 6. The isolated phosphoprotein is acid stable. The trichloroacetic acid denatured 32P-labelled membrane complex is dephosphorylated by hydroxylamine, which might indicate that the phosphorylated protein is an acyl-phosphate. 7. Polyacrylamide gel elctrophoresis (performed with phenol/acetic acid/water) of phosphorylated sarcoplasmic reticulum fractions demonstrates that the 32P-incorporation occurs into a protein of about 100000 molecular weight. 8. It is suggested that the phosphoprotein represents a phosphorylated intermediate of the calcium-dependent ATPase which formation occurs as an early step in the reaction sequence of calcium translocation by cardiac sarcoplasmic reticulum similar as in skeletal muscle.     

The mechanism of glucose 6-phosphate transport by Escherichia coli

To evaluate anion exchange as the mechanistic basis of sugar phosphate transport, natural and artificial membranes were used in studies of glucose 6-phosphate (Glc-6-P) and inorganic phosphate (Pi) accumulation by the uhpT-encoded protein (UhpT) of Escherichia coli. Experiments with intact cells demonstrated that UhpT catalyzed the neutral exchange of internal and external Pi, and work with everted as well as right-side-out membrane vesicles showed further that UhpT mediated the heterologous exchange of Pi and Glc-6-P. When loaded with Pi, but not when loaded with morpholinopropanesulfonate (MOPS), everted vesicles took up Glc-6-P to levels 100-fold above medium concentration in a reaction unaffected by the ionophores valinomycin, valinomycin plus nigericin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similarly, right-side-out vesicles were capable of Glc-6-P transport, but only if a suitable internal countersubstrate was available. Thus, in MOPS-loaded vesicles, oxidative metabolism established a proton-motive force that supported proline or Pi accumulation, but transport of Glc-6-P was found only if vesicles could accumulate Pi during a preincubation. After reconstitution of UhpT into proteoliposomes it was possible to show as well that the level of accumulation of Glc-6-P (17 to 560 nmol/mg of protein) was related directly to the internal concentration of Pi. These results are most easily understood if the transport of glucose 6-phosphate in E. coli occurs by anion exchange rather than by nH+/anion support.