Fructose 2,6-bisphosphate and its phosphorothioate analogue. Comparison of their hydrolysis and action on glycolytic and gluconeogenic enzymes.

Purified chicken liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was phosphorylated either from fructose 2,6-bis[2-32P]phosphate or fructose 2-phosphoro[35S]thioate 6-phosphate. The turnover of the thiophosphorylated enzyme intermediate as well as the overall phosphatase reaction was four times faster than with authentic fructose 2,6-bisphosphate. Fructose 2-phosphorothioate 6-phosphate was 10-100-fold less potent than authentic fructose 2,6-bisphosphate in stimulating 6-phosphofructo-1-kinase and pyrophosphate:fructose 6-phosphate phosphotransferase, but about 10 times more potent in inhibiting fructose 1,6-bisphosphatase. The analogue was twice as effective as authentic fructose 2,6-bisphosphate in stimulating pyruvate kinase from trypanosomes.     

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Summary Fructose was shown to be phosphorylated by a specific phosphoenolpyruvatc-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose Ell. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.     

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.     

Identification of a phosphoenolpyruvate:fructose phosphotransferase system (fructose-1-phosphate forming) in Listeria monocytogenes.

Listeria monocytogenes is a gram-positive bacterium whose carbohydrate metabolic pathways are poorly understood. We provide evidence for an inducible phosphoenolpyruvate (PEP):fructose phosphotransferase system (PTS) in this pathogen. The system consists of enzyme I, HPr, and a fructose-specific enzyme II complex which generates fructose-1-phosphate as the cytoplasmic product of the PTS-catalyzed vectorial phosphorylation reaction. Fructose-1-phosphate kinase then converts the product of the PTS reaction to fructose-1,6-bisphosphate. HPr was shown to be phosphorylated by [32P]PEP and enzyme I as well as by [32P]ATP and a fructose-1,6-bisphosphate-activated HPr kinase like those found in other gram-positive bacteria. Enzyme I, HPr, and the enzyme II complex of the Listeria PTS exhibit enzymatic cross-reactivity with PTS enzyme constituents from Bacillus subtilis and Staphylococcus aureus.     

Pyrophosphate: fructose 6-phosphate phosphotransferase in germinating castor bean seedlings

The distribution of enzymes interconverting fructose 6-phosphate and fructose 1,6-bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PP_i:fructose 6-phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6-bisphosphatase. PP_i:fructose 6-phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.     

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.     

Glycolysis at the climacteric of bananas.

This work was carried out to investigate the relative roles of phosphofructokinase and pyrophosphate-fructose-6-phosphate 1-phosphotransferase during the increased glycolysis at the climacteric in ripening bananas (Musa cavendishii Lamb ex Paxton). Fruit were ripened in the dark in a continuous stream of air in the absence of ethylene. CO2 production, the contents of glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate and PPi; and the maximum catalytic activities of pyrophosphate-fructose-6-phosphate 1-phosphotransferase, 6-phosphofructokinase, pyruvate kinase and phosphoenolpyruvate carboxylase were measured over a 12-day period that included the climacteric. Cytosolic fructose-1,6- bisphosphatase could not be detected in extracts of climacteric fruit. The peak of CO2 production was preceded by a threefold rise in phosphofructokinase, and accompanied by falls in fructose 6-phosphate and glucose 6-phosphate, and a rise in fructose 1,6-bisphosphate. No change in pyrophosphate-fructose-6-phosphate 1-phosphotransferase or pyrophosphate was found. It is argued that phosphofructokinase is primarily responsible for the increased entry of fructose 6-phosphate into glycolysis at the climacteric.     

Role of the phosphoenolpyruvate-dependent fructose phosphotransferase system in the utilization of mannose by Escherichia coli.

Mutants of Escherichia coli devoid of the membrane-spanning proteins PtsG and PtsMP, which are components of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and which normally effect the transport into the cells of glucose and mannose, do not grow upon or take up either sugar. Pseudorevertants are described that take up, and grow upon, mannose at rates strongly dependent on the mannose concentration in the medium (apparent Km > 5 mM); such mutants do not grow upon glucose but are derepressed for the components of the fructose operon. Evidence is presented that mannose is now taken up via the fructose-PTS to form mannose 6-phosphate, which is further utilized for growth via fructose 6-phosphate and fructose 1,6-bisphosphate.     

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。     

Purification and properties of fructose-1,6-bisphosphatase of Bacillus subtilis.

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrase, EC 3.1.3.11) of Bacillus subtilis is a constitutive enzyme that was purified 1000-fold (30% yield) to 80% purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis where it exhibits a band corresponding to 72,000 daltons. It sediments at 15 S in sucrose density gradients indicating a molecular weight of 380,000, but apparently is very asymmetric. Its activity is irreversibly inactivated in the absence of Mn2+. The enzyme specifically catalyzes dephosphorylation of D-fructose 1,6-bisphosphate with a pH optimum of 8.0. It has 40 to 60% of full activity in the absence of P-enolpyruvate; 20 microM P-enolpyruvate activates it maximally. High concentrations of monovalent cations also activate, NH4+ being most effective. Inhibitors fall into two groups. 1) Nucleoside monophosphates, phosphorylated coenzymes, and polynucleotides inhibit competitively with P-enolpyruvate (AMP (Ki = 2 microM) and dAMP are most effective). 2) The inhibition by nucleoside di- and triphosphates, PPi, and highly phosphorylated nucleotides (guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and adenosine 5'-triphosphate 3'-diphosphate are most effective) is not competed by P-enolpyruvate but is partially overcome by fructose 1,6-bisphosphate (2 microM). Therefore, highly phosphorylated nucleotides (pppGpp and others), produced in over 0.2 mM concentrations upon step down from fast to slow growth rates (Gallant, J., and Lazzarini, R.A. (1976) in Protein Synthesis (McConkey, E.H., ed) Vol. 2, pp. 309-349, Marcel Dekker, Inc., New York), can reduce the conversion rate of fructose 1,6-bisphosphate to fructose 6-phosphate during gluconeogenesis. Comparing glycolytic growth on D-glucose and gluconeogenic growth on L-malate, the intracellular concentrations of fructose 1,6-bisphosphate differ but are both above the Km (13 microM) of the enzyme, those of AMP are similar, whereas those of P-enolpyruvate (0.18 mM versus 1.3 mM) indicate that the enzyme has only 40% of its full activity during glycolysis; nucleotides other than AMP may inhibit additionally. Thus, the futile cycle of fructose 1,6-bisphosphate synthesis and degradation during glycolysis is partially avoided, but the cells are poised for rapid adaptation upon change to gluconeogenic growth conditions.     

Glucose 6-Phosphate and Fructose 1,6-Bisphosphate Can Be Used as ATP-Regenerating Systems by Cerebellum Ca2+-Transport ATPase

Abstract : In this work, it is shown that the Ca²⁺-transport ATPase found in the microsomal fraction of the cerebellum can use both glucose 6-phosphate/hexokinase and fructose 1,6-bisphosphate/phosphofructokinase as ATP-regenerating systems. The vesicles derived from the cerebellum were able to accumulate Ca²⁺ in a medium containing ADP when either glucose 6-phosphate and hexokinase or fructose 1,6-bisphosphate and phosphofructokinase were added to the medium. There was no Ca²⁺ uptake if one of these components was omitted from the medium. The transport of Ca²⁺ was associated with the cleavage of sugar phosphate. The maximal amount of Ca²⁺ accumulated by the vesicles with the fructose 1,6-bisphosphate system was larger than that measured either with glucose 6-phosphate or with a low ATP concentration and phosphoenolpyruvate/pyruvate kinase. The Ca²⁺ uptake supported by glucose 6-phosphate was inhibited by glucose, but not by fructose 6-phosphate. In contrast, the Ca²⁺ uptake supported by fructose 1,6-bisphosphate was inhibited by fructose 6-phosphate, but not by glucose. Thapsigargin, a specific SERCA inhibitor, impaired the transport of Ca²⁺ sustained by either glucose 6-phosphate or fructose 1,6-bisphosphate. It is proposed that the use of glucose 6-phosphate and fructose 1,6-bisphosphate as an ATP-regenerating system by the cerebellum Ca²⁺-ATPase may represent a salvage route used at early stages of ischemia ; this could be used to energize the Ca²⁺ transport, avoiding the deleterious effects derived from the cellular acidosis promoted by lactic acid.     

Assessment of a futile cycle involving reconversion of fructose 6-phosphate to fructose 1,6-bisphosphate during gluconeogenic growth of Escherichia coli.

In gluconeogenesis, fructose 6-phosphate is formed from fructose 1,6-bisphosphate, and if fructose 1,6-bisphosphate were reformed by the phosphofructokinase reaction there would be a "gluconeogenic futile cycle." We assessed the extent of this cycling in Escherichia coli growing on glycerol 3-phosphate, using a medium containing 32Pi. Fructose 1,6-bisphosphate coming from glycerol 3-phosphate should be unlabeled, but any coming from fructose 6-phosphate should contain label from the gamma-position of ATP. The amount of labeling of the 1-position of fructose 1,6-bisphosphate was only 2 to 10% of that of the gamma-position of ATP in a series of isogenic strains differing in phosphofructokinases (Pfk-1, Pfk-2, or Pfk-2). In control experiments with glucose 6-phosphate instead of glycerol 3-phosphate, the two positions were equally labeled. Thus, although the presence of Pfk-2 causes gluconeogenic impairment (Daldal et al., Eur. J. Biochem., 126:373-379, 1982), gluconeogenic futile cycling cannot be the reason.     

Ribose 1,5-bisphosphate is a putative regulator of fructose 6-phosphate/fructose 1,6-bisphosphate cycle in liver

6-Phosphofructo-1-kinase and fructose-1,6-bisphosphatase are rate-limiting enzymes for glycolysis and gluconeogenesis respectively, in the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver. The effect of ribose 1,5-bisphosphate on the enzymes was investigated. Ribose 1,5-bisphosphate synergistically relieved the ATP inhibition and increased the affinity of liver 6-phosphofructo-1-kinase for fructose 6-phosphate in the presence of AMP. Ribose 1,5-bisphosphate synergistically inhibited fructose-1,6-bisphosphatase in the presence of AMP. The activating effect on 6-phosphofructo-1-kinase and the inhibitory effect on fructose-1,6-bisphosphatase suggest ribose 1,5-bisphosphate is a potent regulator of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in the liver.     

Compartmentalized ATP synthesis in skeletal muscle triads.

Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6-bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads.     

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.     

Lack of glucose phosphotransferase function in phosphofructokinase mutants of Escherichia coli.

Phosphofructokinase (pfkA) mutants of Escherichia coli are impaired in growth on all carbon sources entering glycolysis at or above the level of fructose 6-phosphate (nonpermissive carbon sources), but growth is particularly slow on sugars, such as glucose, which are normally transported and phosphorylated by the phosphoenolpyruvate, (PEP)-dependent phosphotransferase system (PTS).     

Bioluminescent determination of free fatty acids

A simple, highly specific, and sensitive bioluminescent method for determination of free fatty acids in unextracted plasma or serum has been developed. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The pyrophosphate formed is used to phosphorylate fructose 6-phosphate in a reaction catalyzed by the enzyme pyrophosphate-fructose-6-phosphate phosphotransferase (EC 4.1.2.13). The triosephosphates produced from fructose 1,6-bisphosphate by aldolase are oxidized by NAD in the presence of arsenate to 3-phosphoglycerate. The NADH is detected via the bacterial NADH-linked luciferase system. Excellent agreement has been obtained by comparison with accepted methods. In addition, for the determination of serum free fatty acids, the method is particularly applicable for following lipolysis of isolated adipocytes.     

In vivo regulation of glycolysis and characterization of sugar: phosphotransferase systems in Streptococcus lactis.

Two novel procedures have been used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in Streptococcus lactis ML3. In the first procedure, glucose metabolism was specifically inhibited by p-chloromercuribenzoate. Autoradiographic and enzymatic analyses showed that the cells contained glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-diphosphate, and triose phosphates.Dithiothreitol reversed the p-chloromercuribenzoate inhibition, and these intermediates were rapidly and quantitatively transformed into 3- and 2-phosphoglycerates plus PEP. The three intermediates were not further metabolized and constituted the intracellular PEP potential. The second procedure simply involved starvation of the organisms. The starved cells were devoid of glucose 6-phosphate, fructose 6-phosphate, fructose- 1,6-diphosphate, and triose phosphates but contained high levels of 3- and 2-phosphoglycerates and PEP (ca. 40 mM in total). The capacity to regulate PEP formation in vivo permitted the characterization of glucose and lactose phosphotransferase systems in physiologically intact cells. Evidence has been obtained for "feed forward" activation of pyruvate kinase in vivo by phosphorylated intermediates formed before the glyceraldehyde-3-phosphate dehydrogenase reaction in the glycolytic sequence. The data suggest that pyruvate kinase (an allosteric enzyme) plays a key role in the regulation of glycolysis and phosphotransferase system functions in S. lactis ML3.     

Effects of fructose 1,6-bisphosphate on the activation of yeast phosphofructokinase by fructose 2,6-bisphosphate and AMP

Fructose 1,6-bisphosphate decreases the activation of yeast 6-phosphofructokinase (ATP:fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) by fructose 2,6-bisphosphate, especially at cellular substrate concentrations. AMP activation of the enzyme is not influenced by fructose 1,6-bisphosphate. Inorganic phosphate increases the activation by fructose 2,6-bisphosphate and augments the deactivation of the fructose 2,6-bisphosphate activated enzyme by fructose 1,6-bisphosphate. Because various states of yeast glucose metabolism differ in the levels of the two fructose bisphosphates, the observed interactions might be of regulatory significance.