PfkA locus of Escherichia coli.

pfkA was know, on the basis of three mutants, as the likely locus of phosphofructokinase in Escherichia coli, and the unlinked pfkB1 mutation suppressed these mutations by restoring some enzyme activity (Morrissey and Fraenkel, 1972). We now report a new search for the complete inactivation of pfkA (e.g., by deletion or amber mutation), done to assess whether the pfkB1 suppression is by an independent enzyme, phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973). Ten new phosphofructokinase mutants all were at pfkA, rather than at pfkB or pfkC. One of them (pfkA9) gave temperature-sensitive reverants with heat-labile enzyme. Another (pfkA11) proved genetically to be a nonsense mutation, but showed no restored activity when suppressed by supF. However, even unsuppressed it was found to contain an enzyme related to phosphofructokinase activity 1 kinetically (more allosteric), physically (almot identical subunit), and antigenically. All the pfkA mutants apparently contained cross-reacting material to activity 1. All (including pfkA11) were suppressed by the pfkB1 mutation. Several results support the idea that pfkA is the structural gene for the main phosphofructokinase of E. coli (activity 1), but that there is some restriction to its complete inactivation.     

PfkB and pfkC loci of Escherichia coli.

Mutants lacking Escherichia coli phosphofructokinase (pfkA, 78 min) are suppressed by the unlinked pfkB1 mutation, which restores some enzyme activity (Morrissey and Fraenkel, 1972). We here describe a secondary mutation at pfkB, "PFKB-," which abolishes the suppression as well as the low residual activity of unsuppressed pfkA mutants. pfkB is at about 33 min. with the gene order groD-pps-pheS-pfkB. A positive selection was found that yielded both the pfkB-mutations and a new similar mutation, pfkC-. pfkC is an early marker in Hfr HL16(ca. 50 to 55 min). Some pfkC-, but no pfkB-, mutations were amber. A temperature-sensitive pfkB- was also obtained. Strains carrying pfkB- or pfkC-, but wild type at pfkA, were not markedly affected in growth on sugars. A new search for suppressors such as pfkB1 gave five independent candidates, all of which suppressed both pfkA1 and pfkA2 and occurred in the pfkB region; none occurred at pfkC. Neither the pfkB nor the pfkC loci have assigned functions. It is likely that they are somehow involved in expression of phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973).     

Tn10 insertions in the pfkB region of Escherichia coli.

The locus pfkB is known to determine expression of a minor phosphofructokinase (Pfk-2). Pfk-2 and pfkB seem to be dispensable, since Tn10 insertions in pfkB, as well as deletions from Tn10 nearby, are obtainable. Strains deleted for both pfkA and pgkB are unable to grow at all on sugars whose primary route of metabolism is via fructose 6-phosphate, confirming earlier reports implicating the low Pfk-2 activity, rather than the pentose-phosphate pathway, as needed for the slow growth on sugars of pfkA pfkB+ strains. The pfkB locus probably contains the structural gene for Pfk-2, since a mutation closely linked to pfkB1, which affects growth on glycerol, is found to alter the enzyme. Partial phenotypic suppression of the pfkA mutant phenotype results from Tn10 insertion very close to the pps gene, ca. 0.5 min from pgkB. The insertion does not clearly affect either Pfk-2 or phosphoenolpyruvate synthetase, and the mechanism of suppression is unclear.     

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).     

Oscillatory synthesis of glucose 1,6-bisphosphate and frequency modulation of glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity, due to autocatalytic activation by fructose-1,6-P2. Glucose-1,6-P2 similarly might activate phosphofructokinase in an autocatalytic manner, because it is produced in a side reaction of phosphofructokinase and in a side reaction of phosphoglucomutase using fructose-1,6-P2. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, glucose-1,6-P2 accumulated in a stepwise, but monotonic, manner to 0.7 microM in 1 h. The stepwise increases occurred during the phases when fructose-1,6-P2 was available, consistent with glucose-1,6-P2 synthesis in the phosphoglucomutase side reaction. Addition of 5-20 microM glucose-1,6-P2 increased the frequency of the oscillations in a dose-dependent manner and progressively shortened the time interval before the first burst of phosphofructokinase activity. Addition of 30 microM glucose-1,6-P2 blocked the oscillations. The peak values of the [ATP]/[ADP] ratio were then eliminated, and the average [ATP]/[ADP] ratio was reduced by half. In the presence of higher, near physiological concentrations of ATP and citrate (which reduce the activation of phosphofructokinase by glucose-1,6-P2), high physiological concentrations of glucose-1,6-P2 (50-100 microM) increased the frequency of the oscillations and did not block them. We conclude that autocatalytic activation of phosphofructokinase by fructose-1,6-P2, but not by glucose-1,6-P2, is the mechanism generating the oscillations in muscle extracts. Glucose-1,6-P2 may nevertheless play a role in facilitating the initiation of the oscillations and in modulating their frequency.     

ColE1 hybrid plasmids for Escherichia coli genes of glycolysis and the hexose monophosphate shunt.

The Clarke-Carbon clone bank carrying ColE1-Escherichia coli DNA has been screened by conjugation for complementation of glycolysis and hexose monophosphate shunt mutations. Plasmids were identified for phosphofructokinase (pfkA), triose phosphate isomerase (tpi), phosphoglucose isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf), gluconate-6-phosphate dehydrogenase (gnd), enolase (eno), phosphoglycerate kinase (pgk), and fructose-1,6-P2 aldolase (fda). Enzyme levels for the plasmid-carried gene ranged, for the various plasmids, from 4- to 25-fold the normal level.     

Fructose 2,6-bisphosphate and glycolytic oscillations in skeletal muscle extracts

Oscillatory behavior of glycolysis in cell-free extracts of rat skeletal muscle involves bursts of phosphofructokinase activity due to autocatalytic activation by fructose-1,6-P2. Fructose-2,6-P2 is an even more potent activator of phosphofructokinase and is competitive with fructose-1,6-P2 in binding and kinetic studies. The possible role and effects of fructose-2,6-P2 on the oscillating system were therefore examined. When muscle extracts were provided with 1 mM ATP and 10 mM glucose, fructose-2,6-P2 slowly accumulated to 50 nM in 1 h. The nearly monotonic rise, in contrast to the 50-fold oscillations in fructose-1,6-P2, indicated no involvement of fructose-2,6-P2 in the oscillatory process. Addition of 0.5 microM fructose-2,6-P2 blocked the oscillations, and there was negligible appearance of glycolytic intermediates from fructose-1,6-P2 to phosphoenolpyruvate, although similar amounts of lactate accumulated. In the presence of 0.2 microM fructose-2,6-P2, there were small, transient accumulations of fructose-1,6-P2, suggesting aborted activations of phosphofructokinase. Oscillations were not blocked by 0.1 microM fructose-2,6-P2. The average [ATP]/[ADP] ratio in the presence of 0.2 or 0.5 microM fructose-2,6-P2 was half the value in its absence, demonstrating the advantage of the oscillatory behavior in maintaining a high energy state. In the presence of higher, near physiological levels of ATP and citrate, inhibitors which reduce the affinity of phosphofructokinase for fructose-2,6-P2, glycolytic oscillations were not blocked by 1 microM fructose-2,6-P2, its approximate concentration in vivo.     

Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. Mutation at the substrate-binding site affects allosteric behavior

Arg252 of fructose-6-phosphate 1-kinase (PFK) from Bacillus stearothermophilus has been proposed to be involved in the binding of the substrate Fru-6-P. We demonstrate here that mutation of this residue to alanine converts the enzyme to a form with characteristics similar to those of its allosterically tight form. The mutant enzyme exhibits a high affinity for its inhibitor phosphoenolpyruvate (a 68-fold difference compared to wild type) and a dramatically decreased Fru-6-P affinity (1500-fold increase in Km). It is more sensitive to inhibition by high ATP concentrations than the wild type, and this inhibition is relieved by ADP, GDP, or higher Fru-6-P concentrations. In contrast, mutation of Arg252 to lysine increases the affinity of the enzyme for P-enolpyruvate by only 2-fold and increases its Km for Fru-6-P by only 50-fold. Sigmoidal kinetics with respect to Fru-6-P in the presence of P-enolpyruvate were observed with Hill numbers of 2.2, 2.4, and 1.7 for wild-type B. stearothermophilus PFK and the Arg252 to lysine and to alanine mutations, respectively. Unlike fructose-6-phosphate 1-kinase from Escherichia coli, in the absence of P-enolpyruvate, B. stearothermophilus PFK exhibits a hyperbolic profile with respect to Fru-6-P concentration. B. stearothermophilus PFK is sensitive to inhibition by high ATP concentrations and competitively inhibited by GDP or ADP. Our data indicate that Arg252 of B. stearothermophilus PFK plays a major role in both Fru-6-P binding and allosteric interaction between the subunits. However, this residue does not seem to participate directly in the catalytic process.     

Metabolism of glucose 1,6-P2. I. Enzymes involved in the synthesis of glucose 1,6-P2 in pig tissues

Pig tissues show four enzymatic activities of glucose 1,6-P2 synthesis: (A) 2 [glucose 1-P]----glucose 1,6-P2 + glucose; (B) glucose 1-P + ATP----glucose 1,6-P2 + ADP; (C) glucose 1-P + fructose 1,6-P2----glucose 1,6-P2 + fructose 6-P; (D) glucose 1-P + glycerate 1,3-P2----glucose 1,6-P2 + glycerate 3-P. Brain is the tissue with highest capability of glucose 1,6-P2 synthesis. With the exception of skeletal muscle, activity "D" represents the highest activity of glucose 1,6-P2 synthesis. In muscle, activity "B" is the major activity. The existence of a specific glucose 1,6-P2 synthase which catalyzes reaction "D" is confirmed. Two peaks of such an enzyme are isolated by ion-exchange chromatography. There is an enzyme which specifically catalyzes reaction "C", not previously described. There is a glucose 1-P kinase not identical to phosphofructokinase.     

A specific enzyme for glucose 1,6-bisphosphate synthesis.

The reaction: glycerate-1,3-P2 PLUS GLUCOSE-1-P YIELDS TO GLUCOSE-1,6-P2 plus glycerate-P is catalyzed by a distinct enzyme of mouse brain. A divalent metal requirement was shown when the enzyme was treated with imidazole and EDTA. Mg2+, Mn2+, Ca2+, Zn2+, Ni2+, Co2+, and Cd2+ were quite effective cofactors. The enzyme, in better than 50 percent yield, has been purified away from 99 percent of the phosphoglucomutase, phosphoglycrate mutase, and phosphofructokinase. Acetyl-P, ATP, enolpyruvate-P, creatine-P, and fructose-1,6-P2 are not phosphoryl donors. Glucose-6-P and mannose-1-P are good alternate acceptors. Mannose-6-P, galactose-Ps, and fructose-Ps have little or no acceptor activity. Strong inhibition was found with fructose-1,6-P2, glycerate-2,3-P2, enolpyruvate-P, and acetyl CoA. From the amount of activity and the kinetic constants of the purified enzyme it seems likely that this enzyme is responsible for the glucose-1,6-P2 synthesis of brain.     

Chimeric phosphofructokinases involving exchange of the N- and C-terminal halves of mammalian isozymes: implications for ligand binding sites

Two phosphofructokinase (PFK) chimeras were constructed by exchanging the N- and C-terminal halves of the mammalian M- and C-type isozymes, to investigate the contribution of each terminus to the catalytic site and the fructose-2,6-P(2)/fructose-1,6-P(2) allosteric site. The homogeneously-purified chimeric enzymes organized into tetramers, and exhibited kinetic properties for fructose-6-P and MgATP similar to those of the native enzyme that furnished the N-terminal domain in each case, whereas their fructose-2,6-P(2) activatory characteristics coincided with those of the isozyme that provided the C-terminal half. This reflected the role of each domain in the formation of the corresponding binding site. Grafting the N-terminus of PFK-M onto the C-terminus of the fructose-1,6-P(2) insensitive PFK-C restored transduction of this signal to the catalytic site, which significance is also discussed.     

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.     

Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the expression and purification of phosphofructokinase from bacterial sources

As the key obligatory step in the glycolytic pathway, the regulation of phosphofructokinase (PFK-1) has been the focus of study of several laboratories. While standard cloning procedures have opened the door to study PFK from a vast array of sources, a good pfk knockout Escherichia coli strain has not previously been developed. Many laboratories rely on DF1020 or similar derivatives for PFK expression. Unfortunately, DF1020 grows poorly and does not have an inherent means for controlling expression of genes from plasmids. More importantly, however, DF1020 has a tendency to grow on minimal media when glucose is used as the sole carbon source. In this study, a new E. coli PFK expression strain lacking both PFK-1 and PFK-2 has been engineered using lambda-red mediated chromosomal deletion. The resulting strain has been designated RL257. In addition to having both pfkA and pfkB deleted, RL257 contains the lacI(q) allele, which allows for inducible expression when coupled with an expression vector containing either the lac or tac promoter.     

Phosphofructokinase C isozyme from ascites tumor cells: cloning, expression, and properties

The phosphofructokinase C isozyme (PFK-C) from ascites tumor cells has been cloned and characterized to investigate the particular properties of PFK activity in this type of cells. The isolated cDNA encodes a protein of 784 amino acids and 85.5 kDa, whose expression was constant along tumor growth and markedly decreased when cell proliferation stops. The enzyme was functionally expressed in a PFK-deficient strain of Saccharomyces cerevisiae and purified to homogeneity. Recombinant PFK-C exhibited the same subunit size as the tumor wild-type isozyme and its steady-state kinetic parameters were similar to those of the form present in normal cells. The regulatory properties of the C isozyme accounted for the lack of fructose-1,6-P(2) activation and the P-enolpyruvate inhibition of PFK activity observed in ascites tumor preparations containing the various isozyme types. Nevertheless, PFK-C binds fructose-1,6-P(2) to an allosteric site as suggested by protection against thermal denaturation. Our results indicate that glucose metabolism in tumor cells is not regulated by a mutant form of PFK-C but by a high level expression of the normal C isozyme.     

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.     

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.     

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.     

The role of phosphofructokinase in glycolytic control in the facultative anaerobe Sipunculus nudus

Summary The involvement of phosphofructokinase (PFK) in glycolytic control was investigated in the marine peanut worm Sipunculus nudus. Different glycolytic rates prevailed at rest and during functional and environmental anaerobiosis: in active animals glycogen depletion was enhanced by a factor of 120; during hypoxic exposure the glycolytic flux increased only slightly. Determination of the mass action ratio (MAR) revealed PFK as a non-equilibrium enzyme in all three physiological situations. Duirng muscular activity the PFK reaction was shifted towards equilibrium; this might account for the observed increase in glycolytic rate under these conditions. PFK was purified from the body wall muscle of S. nudus. The enzyme was inhibited by physiological ATP concentrations and an acidic pH; adenosine monophosphate (AMP), inorganic phosphate (P_i), and fructose-2,6-bisphosphate (F-2,6-P₂) served as activators. PFK activity, determined under simulated cellular conditions of rest and muscular work, agreed well with the glycolytic flux in the respective situations. However, under hypoxia PFK activity surpassed the glycolytic rate, indicating that PFK may not be rate-limiting under these conditions. The results suggest that glycolytic rate in S. nudus is mainly regulated by PFK during rest and activity. Under hypoxic conditions the regulatory function of PFK is less pronounced.Abbreviations ATP, ADP, AMP adenosine tri-, di-, monophosphate - DTT dithiothreitol - EDTA ethylene diaminetetra-acetic acid - F-6-P fructose-6-phosphate - F-1,6-P₂ fructose-1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate; bwm, body wall muscle; fresh mass, total body weight - G-6-P glucose-6-phosphate - H enthalpy change - K _a activation constant - K _eq equilibrium constant - K _i inhibition constant - K _m Michaelis constant - MAR mass action ratio - NMR nuclear magnetic resonance - PFK phosphofructokinase - P_i inorganic phosphate - PLA phospho-l-arginine - SD standard deviation - TRIS, TRIS (hydroxymethyl) aminomethane - TRA triethanolamine hydrochloride - V _max maximal velocity