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Most cancer cells exhibit high glycolysis rates under conditions of abundant oxygen. Maintaining a stable glycolytic rate is critical for cancer cell growth as it ensures sufficient conversion of glucose carbons to energy, biosynthesis, and redox balance. Here we deciphered the interaction between PKM2 and the thermodynamic properties of the glycolytic pathway. Knocking down or knocking out PKM2 induced a thermodynamic equilibration in the glycolytic pathway, characterized by the reciprocal changes of the Gibbs free energy (ΔG) of the reactions catalyzed by PFK1 and PK, leading to a less exergonic PFK1-catalyzed reaction and a more exergonic PK-catalyzed reaction. The changes in the ΔGs of the two reactions cause the accumulation of intermediates, including the substrate PEP (the substrate of PK), in the segment between PFK1 and PK. The increased concentration of PEP in turn increased PK activity in the glycolytic pathway. Thus, the interaction between PKM2 and the thermodynamic properties of the glycolytic pathway maintains the reciprocal relationship between PK concentration and its substrate PEP concentration, by which, PK activity in the glycolytic pathway can be stabilized and effectively counteracts the effect of PKM2 KD or KO on glycolytic rate. In line with our previous reports, this study further validates the roles of the thermodynamics of the glycolytic pathway in stabilizing glycolysis in cancer cells. Deciphering the interaction between glycolytic enzymes and the thermodynamics of the glycolytic pathway will promote a better understanding of the flux control of glycolysis in cancer cells., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3- 3 H]glucose relative to [2- 3 H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2- 13 C 2 ]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, P i , and glycerol 3-phosphate., (© 2020 Moonira et al.)

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C 5 precursors (such as ribose) during growth on other (non-C 5 ) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PP i -PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PP i -PFKs of Clostridium thermosuccinogenes and C lostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli , which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PP i -PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K ½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia., (© 2020 Koendjbiharie et al.)

The mechanisms by which metformin (dimethylbiguanide) inhibits hepatic gluconeogenesis at concentrations relevant for type 2 diabetes therapy remain debated. Two proposed mechanisms are 1) inhibition of mitochondrial Complex 1 with consequent compromised ATP and AMP homeostasis or 2) inhibition of mitochondrial glycerophosphate dehydrogenase (mGPDH) and thereby attenuated transfer of reducing equivalents from the cytoplasm to mitochondria, resulting in a raised lactate/pyruvate ratio and redox-dependent inhibition of gluconeogenesis from reduced but not oxidized substrates. Here, we show that metformin has a biphasic effect on the mitochondrial NADH/NAD redox state in mouse hepatocytes. A low cell dose of metformin (therapeutic equivalent: <2 nmol/mg) caused a more oxidized mitochondrial NADH/NAD state and an increase in lactate/pyruvate ratio, whereas a higher metformin dose (≥5 nmol/mg) caused a more reduced mitochondrial NADH/NAD state similar to Complex 1 inhibition by rotenone. The low metformin dose inhibited gluconeogenesis from both oxidized (dihydroxyacetone) and reduced (xylitol) substrates by preferential partitioning of substrate toward glycolysis by a redox-independent mechanism that is best explained by allosteric regulation at phosphofructokinase-1 (PFK1) and/or fructose 1,6-bisphosphatase (FBP1) in association with a decrease in cell glycerol 3-phosphate, an inhibitor of PFK1, rather than by inhibition of transfer of reducing equivalents. We conclude that at a low pharmacological load, the metformin effects on the lactate/pyruvate ratio and glucose production are explained by attenuation of transmitochondrial electrogenic transport mechanisms with consequent compromised malate-aspartate shuttle and changes in allosteric effectors of PFK1 and FBP1., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Alshawi and Agius.)

It has been recognized that the rate-limiting function of pyruvate kinase M2 (PKM2) in glycolysis plays an important role in distributing glycolytic intermediates for anabolic and catabolic purposes in cancer cells. However, after analysis of the catalytic capacity of PKM2 relative to other glycolytic enzymes, the regulation range of PKM2 activity, metabolic flux control, and thermodynamics, we suggest that the PKM2-catalyzed reaction is not a rate-limiting step in cancer cell glycolysis. Hexokinase and phosphofructokinase 1 (PFK1), the first and third enzyme along the pathway, are rate-limiting enzymes that limit the overall glycolytic rate, whereas PKM2 and lactate dehydrogenase, the last two enzymes in the pathway, are for the fast removal of upstream intermediates to prevent the obstruction of the pathway. The argument is in accordance with the catalytic capacity of glycolytic enzymes, regulation range of enzyme activities, metabolic flux control, and thermodynamics., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pulses of insulin released from pancreatic β-cells maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) enables β-cells to generate metabolic oscillations via autocatalytic activation by its product fructose 1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has been lacking. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; pyruvate kinase activity reporter), which multimerizes and is activated upon binding FBP. When introduced into Min6 β-cells, PKAR FRET efficiency increased rapidly in response to glucose. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, consistent with sensor activation by FBP; the dependence of PKAR on FBP was further confirmed using purified sensor in vitro. Using a novel imaging modality for monitoring mitochondrial flavin fluorescence in mouse islets, we show that slow oscillations in mitochondrial redox potential stimulated by 10 mm glucose are in phase with glycolytic efflux through PKM2, measured simultaneously from neighboring islet β-cells expressing PKAR. These results indicate that PKM2 activity in β-cells is oscillatory and are consistent with pulsatile PFK1 being the mediator of slow glycolytic oscillations.

The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent Ki∼1 μM). In sharp contrast, short chain acyl-CoAs, palmitoylcarnitine, and palmitic acid in the presence of CoASH were without effect. Remarkably, MgAMP and MgADP but not MgATP protected PFK-1 against inhibition by palmitoyl-CoA indicating that acyl-CoAs regulate PFK-1 activity in concert with cellular high energy phosphate status. Furthermore, incubation of PFK-1 with [1-(14)C]palmitoyl-CoA resulted in robust acylation of the enzyme that was reversible by incubation with acyl-protein thioesterase-1 (APT1). Importantly, APT1 reversed palmitoyl-CoA-mediated inhibition of PFK-1 activity. Mass spectrometric analyses of palmitoylated PFK-1 revealed four sites of acylation, including Cys-114, Cys-170, Cys-351, and Cys-577. PFK-1 in both skeletal muscle extracts and in purified form was inhibited by S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, demonstrating that covalent acylation of PFK-1 was not required for inhibition. Tryptic footprinting suggested that S-hexadecyl-CoA induced a conformational change in PFK-1. Both palmitoyl-CoA and S-hexadecyl-CoA increased the association of PFK-1 with Ca2+/calmodulin, which attenuated the binding of palmitoylated PFK-1 to membrane vesicles. Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K(0.5) for fructose-6-P and a decrease in the apparent k(cat) as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n(H) of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.

The mitogen-activated protein kinase JNK1 suppresses interleukin-3 withdrawal-induced cell death through phosphorylation of the BH3-only pro-apoptotic Bcl-2 family protein Bad at Thr-201. It is unknown whether JNK1 regulates glycolysis, an important metabolic process that is involved in cell survival, and if so, whether the regulation depends on Thr-201 phosphorylation of Bad. Here we report that phosphorylation of Bad by JNK1 is required for glycolysis through activation of phosphofructokinase-1 (PFK-1), one of the key enzymes that catalyze glycolysis. Genetic disruption of Jnk1 alleles or silencing of Jnk1 by small interfering RNA abrogates glycolysis induced by growth/survival factors such as serum or interleukin-3. Proteomic analysis identifies PFK-1 as a novel Bad-associated protein. Although the interaction between PFK-1 and Bad is independent of JNK1, Thr-201 phosphorylation of Bad by JNK1 is required for PFK-1 activation. Thus, our results provide a novel molecular mechanism by which JNK1 promotes glycolysis for cell survival.

Here we describe the crystal structures of the NAD kinase (LmNADK1) from Listeria monocytogenes in complex with its substrate NAD, its product NADP, or two synthesized NAD mimics. We identified one of the NAD mimics, di-adenosine diphosphate, as a new substrate for LmNADK1, whereas we showed that the closely related compound di-5'-thioadenosine is a novel non-natural inhibitor for this enzyme. These structures suggest a mechanism involving substrate-assisted catalysis. Indeed, sequence/structure comparison and directed mutagenesis have previously shown that NAD kinases (NADKs) and the distantly related 6-phosphofructokinases share the same catalytically important GGDGT motif. However, in this study we have shown that these enzymes use the central aspartate of this motif differently. Although this acidic residue chelates the catalytic Mg(2+) ion in 6-phosphofructokinases, it activates the phospho-acceptor (NAD) in NADKs. Sequence/structure comparisons suggest that the role of this aspartate would be conserved in NADKs and the related sphingosine and diacylglycerol kinases.

Trypanosoma brucei and related organisms contain an organelle evolutionarily related to peroxisomes that sequesters glycolysis, among other pathways. We have shown previously that disruption of protein import into this organelle, the glycosome, can be accomplished through RNA interference (RNAi)-mediated knockdown of the peroxin PEX14. Decreased PEX14 in turn leads to cell death, which, at least in the procyclic stage, can be triggered by the presence of glucose. Here we show that fructose, which is taken up and metabolized by procyclic form T. brucei, and glycerol, which interfaces with the glycosomal glycolytic pathway, are also toxic during PEX14 RNAi. Earlier computer modeling studies predicted that glycolysis would be toxic to T. brucei in the absence of glycosomal compartmentation because of the intrinsic lack of feedback regulation of the parasite hexokinase and phosphofructokinase. To further test this hypothesis, we performed double RNAi, targeting hexokinase and PEX14. Knockdown of hexokinase rescued PEX14 knockdown cells from glucose toxicity, even though glycosomal proteins continue to be mislocalized to the cytosol. Knockdown of phosphofructokinase was benign in the absence of glucose but toxic in the presence of glucose. When PEX14 and phosphofructokinase mRNAs were jointly targeted for RNAi, glycerol remained toxic to the parasites. Taken together, these data indicate that the glycosome provides significant, but not complete, protection of trypanosomes from the dangerous design of glycolysis.

Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.

Two phosphofructokinase genes have been described previously in Entamoeba histolytica. The product of the larger of the two genes codes for a 60-kDa protein that has been described previously as a pyrophosphate (PP(i))-dependent enzyme, and the product of the second, coding for a 48-kDa protein, has been previously reported to be a PP(i)-dependent enzyme with extremely low specific activity. Here it is found that the 48-kDa protein is not a PP(i)-dependent enzyme but a highly active ATP-requiring enzyme (k(cat) = 250 s(-)1) that binds the cosubstrate fructose 6-phosphate (Fru-6-P) with relatively low affinity. This enzyme exists in concentration- and ATP-dependent tetrameric active and dimeric inactive states. Activation is achieved in the presence of nucleoside triphosphates, ADP, and PP(i), but not by AMP, P(i), or the second substrate Fru-6-P. Activation by ATP is facilitated by conditions of molecular crowding. Divalent cations are not required, and no phosphoryl transfer occurs during activation. Kinetics of the activated enzyme show cooperativity with Fru-6-P (Fru-6-P(0.5) = 3.8 mm) and inhibition by high ATP and phosphoenolpyruvate. The enzyme is active without prior activation in extracts of E. histolytica. The level of mRNA, the amount of enzyme protein, and the enzyme activity of the 48-kDa enzyme are about one-tenth that of the 60-kDa enzyme in extracts of E. histolytica trophozoites.

Yeast phosphofructokinase is a heterooctameric enzyme subject to a complex allosteric regulation. A mutation in the PFK1 gene, encoding the larger -subunits, rendering the enzyme insensitive to allosteric inhibition by ATP was found to be caused by an exchange of proline 728 for a leucine residue. By in vitro mutagenesis, we introduced this mutation in either PFK1 or PFK2 and found that the exchange in either subunit drastically reduced the sensitivity of the holoenzyme to ATP inhibition. This was accompanied by a lack of allosteric activation by AMP, fructose 2,6-bisphosphate, or ammonium and an increased resistance to heat inactivation. Yeast cells carrying either one mutation or both in conjunction did not display a strong phenotype when grown on fermentable carbon sources and did not show any significant changes in intermediary metabolites. Growth on non-fermentable carbon sources was clearly impaired. The strain carrying both mutant alleles was more sensitive to Congo Red than the wild-type strain or the single mutants indicating differences in cell wall composition. In addition, we found single pfk null mutants to be less viable than wild type at different storage temperatures and a pfk2 null mutant to be temperature-sensitive for growth at 37 degrees C. The latter mutant was shown to be respiration-dependent for growth on glucose.

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.

Neurons that express neuronal nitric-oxide synthase (nNOS) are resistant to NO-induced neurotoxicity; however, the mechanism by which these neurons are protected is not clear. To identify proteins possibly involved in this process, we performed affinity chromatography with the nNOS PDZ domain, a N-terminal motif that mediates protein interactions. Using this method to fractionate soluble tissue extracts, we identified the muscle isoform of phosphofructokinase (PFK-M) as a protein that binds to nNOS both in brain and skeletal muscle. PFK-M interacts with the PDZ domain of nNOS, and nNOS-PFK-M binding can be competed by peptides that bind to the PDZ domain of nNOS. We found that nNOS is significantly associated with PFK-M in skeletal muscle because nNOS can be immunodepleted from cytosolic skeletal muscle extracts using an antibody directed against PFK-M. In brain, nNOS and PFK-M are both enriched in synaptosomes, and specifically, in the synaptic vesicle fraction, where they can interact. At the cellular level, PFK-M is enriched in neurons that express nNOS protein. As fructose-1, 6-bisphosphate, the product of PFK activity, is neuroprotective, the interaction of nNOS and PFK may contribute to neuroprotection of nNOS positive cells.

Simultaneous binding of two sequential glycolytic enzymes, phosphofructokinase and aldolase, to a microtubular network was investigated. The binding of the phosphofructokinase to microtubules and its bundling activity has been previously characterized (Lehotzky, A., Telegdi, M., Liliom, K., and Ovádi, J. (1993) J. Biol. Chem. 268, 10888-10894). Aldolase binding to microtubules at near physiological ionic strength is weak (Kd = 20 microM) as compared with that of the kinase (Kd = 1 microM). The interactions of both enzymes with microtubules are modulated by their common intermediate, fructose-1,6-bisphosphate. Pelleting and electron microscopic measurements have revealed that the aldolase binding interferes with that of phosphofructokinase, although they have distinct binding domains on microtubules. The underlying molecular mechanism responsible for this finding is that in the solution phase aldolase and phosphofructokinase form a bienzyme complex that does not bind to the microtubule. The bienzyme complex formation does not influence the catalytic activity of aldolase, however, it inhibits the dissociation-induced inactivation of the kinase by stabilizing a catalytically active molecular form. The present data suggest the first experimental evidence that two sequential glycolytic enzymes do not associate simultaneously to microtubules, but their complexation in solution provides kinetic advantage for glycolysis.

The SNF1 gene encodes a protein kinase necessary for expression of glucose-repressible genes and for the synthesis of the storage polysaccharide glycogen. From a genetic screen, we have found that mutation of the PFK2 gene, which encodes the beta-subunit of 6-phosphofructo-1-kinase, restores glycogen accumulation in snf1 cells. Loss of PFK2 causes elevated levels of metabolites such as glucose-6-P, hyperaccumulation of glycogen, and activation of glycogen synthase, whereas glucose-6-P is reduced in snf1 cells. Other mutations that increase glucose-6-P, deletion of PFK1, which codes for the alpha-subunit of 6-phosphofructo-1-kinase, or of PGI1, the phosphoglucoisomerase gene, had similar effects on glycogen metabolism as did pfk2 mutants. We propose that elevated glucose-6-P mediates the effects of these mutations on glycogen storage. Glycogen synthase kinase activity was reduced in extracts from pfk2 cells but was restored to that of wild type if the extract was gel-filtered to remove small molecules. Also, added glucose-6-P inhibited the glycogen synthase kinase activity in extracts from wild-type cells, half-maximally at approximately 2 mM. We suggest that glucose-6-P controls glycogen synthase activity by two separate mechanisms. First, glucose-6-P is a direct activator of glycogen synthase, and second, it controls the phosphorylation state of glycogen synthase by inhibiting a glycogen synthase kinase.

31P NMR was used to measure the intracellular free magnesium concentration ([Mg2+]i) in human erythrocytes while [Mg2+]i was changed between 0.01 and 1.2 mM using the divalent cationophore A23187. 13C NMR and [2-13C]glucose were used to determine the kinetic effects of [Mg2+]i by measuring the flux through several parts of the glucose pathway. Glucose utilization was strongly dependent on [Mg2+]i, with half-maximal flux occurring at 0.03 mM. The rate-limiting step was most likely at phosphofructokinase, which has a Km(Mg2+) of 0.025 mM in the purified enzyme. Phosphorylated glycolytic intermediate concentration was also strongly dependent on [Mg2+]i and [MgATP], and glucose transport plus hexokinase may have been partially rate-determining at [Mg2+]i below approximately 0.1 mM. The pentose phosphate shunt activity was too low to determine the dependence on [Mg2+]i. Phosphoglycerate kinase and 2, 3-diphosphoglycerate mutase fluxes were also measured, but were not rate-limiting for glycolysis and showed no Mg2+ dependence. Human erythrocyte [Mg2+]i varies between 0.2 mM (oxygenated) and 0.6 mM (deoxygenated), well above the measured [Mg2+]i(1/2). It is unlikely, then, that [Mg2+]i plays a regulatory role in normal erythrocyte glycolysis.

Muscle type phosphofructokinase (M-PFK) deficiency is a rare inherited glycogen storage disease in humans that causes exertional myopathy and hemolysis. The molecular basis of canine M-PFK deficiency, the only naturally occurring animal homologue, was investigated. Lack of M-PFK enzyme activity was caused by a nonsense mutation in the penultimate exon of the M-PFK gene, leading to rapid degradation of a truncated (40 amino acids) and therefore unstable M-PFK protein. A polymerase chain reaction-based test was devised to identify M-PFK-deficient and carrier animals. This represents one of only a few inborn errors of metabolism where the molecular defect has been identified in a large animal model which can now be used to develop and assess novel therapeutic strategies.

In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase. Specifically, we identified two amino acids involved in the binding of the most potent allosteric activator fructose 2,6-bisphosphate. Thus, Ser724 was replaced by an aspartate and His859 by a serine in each of the enzyme subunits. Whereas the substitutions had no drastic effects when introduced only in one of the two types of subunits, kinetic parameters were modified when both subunits carried the mutation. Thus, the enzyme with His859 --> Ser showed an increase in Ka for binding of the activator, whereas the one with Ser724 --> Asp failed to react to the addition of fructose 2, 6-bisphosphate, at all. The enzymes still responded to other allosteric activators, such as AMP. Stabilities of the mutant subunits were not significantly altered in vivo, as judged from Western blot analysis. Phenotypically, strains expressing the mutant PFK genes showed a pronounced effect on the level of intermediary metabolites after growth on glucose. Mutants not responding to the activator at all (Ser724 --> Asp) also displayed higher generation times on glucose medium. This could be suppressed by increasing the gene dosage of the mutant alleles. These results indicate that fructose 2,6-bisphosphate through its activation of phosphofructokinase plays an important role in regulation of the glycolytic flux.

The phosphofructokinases (PFKs) from the bacteria Escherichia coli and Bacillus stearothermophilus differ markedly in their regulation by ATP. Whereas E. coli PFK (EcPFK) is profoundly inhibited by ATP, B. stearothermophilus PFK (BsPFK) is only slightly inhibited. The structural basis for this difference could be closure of the active site via a conformational transition that occurs in the ATP-binding domain of EcPFK, but is absent in BsPFK. To investigate the role of this transition in ATP inhibition of EcPFK, we have constructed a chimeric enzyme that contains the "rigid" ATP-binding domain of BsPFK grafted onto the remainder of the EcPFK subunit. The chimeric PFK has the following characteristics: (i) tetrameric structure and kinetic parameters similar to those of the native enzymes, (ii) insensitivity to regulation by the effector phosphoenolpyruvate despite its ability to bind to the enzyme, and (iii) a sigmoidal (nH around 2) fructose 6-phosphate saturation curve. From the results, it is concluded that the active site regions of the two native enzymes are remarkably similar, but their effector sites and their mechanisms of heterotropic regulation are different. The chimeric subunit is locked in a structure resembling that of activated E. coli PFK, yet the enzyme can exist in two different conformational states. Mechanisms for its sigmoidal kinetics are discussed.

The activity of Escherichia coli phosphofructo-1-kinase depends upon the dissociation of a group with a pK of approximately 6.6. Mutation of the 2 active site residues most likely to be titrated in this range, Asp-127 and Asp-129, lowered activity but produced little change in the pH dependence, suggesting that these residues while important to activity were not responsible for the pH dependence of the enzyme. Alternatively, the pH dependence was thought to correspond to the negative charge on the phosphoryl group being transferred in the reaction. Mutation of Arg-72 to histidine, while lowering the activity substantially, shifted the pH optimum to approximately 6.2 with a secondary plateau on the alkaline limb that was eliminated in the double mutant R72H/R171H. Mutation of Arg-171 alone produced only a small decrease in maximal activity and little change in pH dependence from the wild type enzyme. The data support an associative mechanism with a transition state stabilized by the interaction between the negative charge on the phosphoryl group being transferred and the arginyl residue at position 72 on the enzyme.

To elucidate the structure, tissue-specific expression, and allosteric properties of phosphofructokinase-C (PFK-C), we cloned the cDNA for PFK-C from a rat hypothalamic cDNA library. The cDNA is 2643 base pairs long and encodes a protein of 765 amino acids. The deduced amino acid sequence is highly homologous to PFK-M (muscle) and PFK-L (liver), 69 and 65% amino acid identity, respectively, especially at substrate binding and catalytic sites, while the allosteric binding sites are less conserved. Tissue-specific expression of PFK-C was investigated by Northern blot analysis. PFK-C mRNA was detected in several brain regions and the anterior pituitary but not in liver, skeletal muscle, or several other tissues. In situ hybridization showed that PFK-C is expressed at a higher level in higher brain regions such as the cortex, compared with the midbrain and basal ganglia, while PFK-L is expressed at approximately equal levels throughout the brain. Expression plasmids containing PFK-C and PFK-L coding sequences were constructed and expressed by transient transfection into CMT cells. Expression of transfected PFKs was demonstrated by PFK enzymatic activity and by Western blotting with anti-rat brain and liver PFK antisera. Allosteric regulatory properties of PFK-C and PFK-L expressed in CMT cells were compared. Fructose 2,6-bisphosphate, a potent activator of PFK, decreased the Km of PFK-C for fructose 6-phosphate from 200 to 60 microM while decreasing that of PFK-L from 300 to 55 microM. The properties of PFK-C and PFK-L expressed in CMT cells clearly demonstrate the allosteric differences between the different PFK isozymes.

Two instances, involving the enzymes carbamoyl-phosphate synthetase from Escherichia coli and phosphofructokinase from Bacillus stearothermophilus, respectively, are described in which increasing temperature alone causes the actions of an allosteric ligand to change from inhibition to activation. In neither case are these effects due to a change in the activation energy of the enzyme catalyzed reaction induced by the allosteric ligand. Rather, they are due to temperature-dependent changes in the extent to which the binding of allosteric ligand modifies the affinity of the enzyme for substrate. The data can be readily explained by an analysis of the apparent delta H and delta S components of the coupling free energy, which quantitatively describe the actions of allosteric ligands that act in this manner. These observations underscore the shortcomings of expecting to explain the actions of an allosteric ligand solely by the structural perturbations that accompany the binding of an allosteric ligand such as those often revealed by x-ray crystallography.

Full-length cDNA for rabbit muscle phosphofructokinase has been cloned and characterized (Li, J., Chen, Z., Lu, L., Byrnes, M., and Chang, S. H. (1990) Biochem. Biophys. Res. Commun. 170, 1056-1060). The 2.8-kilobase cDNA was inserted in the plasmid vector pPL2 and transformed into Escherichia coli cells deficient in endogenous phosphofructokinase activity (DF 1020). The recombinant phosphofructokinase so prepared is nearly identical in kinetic properties and size of subunits to the enzyme isolated from rabbit muscle. On the basis of the sequence homology between the muscle and the bacterial phosphofructokinases and the crystallographic structure of the latter, the glutamine at position 200 of the muscle enzyme is implicated in the allosteric transitions. This residue was replaced by alanine (Q200A), glutamate (Q200E), or arginine (Q200R). The purified enzymes were analyzed for quaternary structure, activity, and allosteric properties. The native and all the altered enzymes are tetramers. At pH 7.0, the wild-type enzyme is sensitive to inhibition by ATP at concentration above 0.6 mM, and its activity responds to fructose 6-phosphate concentration cooperatively at high ATP concentration. In contrast, the mutated enzyme Q200R is virtually insensitive to ATP inhibition up to 7 mM. Thus at high ATP concentration, its activity responds to fructose 6-phosphate concentration is a manner similar to the activated form of the native enzyme. Under the same conditions, mutant Q200E exhibits cooperative behavior only at much higher concentration of fructose 6-phosphate. Mutant Q200A is active at pH 8.0 but inactive at pH 7.0. The native enzyme and all three mutants are activated by inorganic phosphate and fructose 2,6-bisphosphate and inhibited by citrate.

Gluconeogenic flux exceeds glycolytic flux at the hexose-phosphate steps when measured in extracts of kidney cortex from well-fed rats. Addition of AMP and/or fructose 2,6-bisphosphate to the assay medium partially eradicates the difference. Using principles developed by Kacser, H., and Burns, J. A. ((1973) in Rate Control of Biological Processes (Davies, D. D., ed) pp. 65-104, Cambridge University Press, London) and Heinrich R., and Rapoport T. A. ((1974) Eur. J. Biochem. 42, 97-105), flux control coefficients of enzymes participating in the pathway segments from glucose 6-phosphate to triose-phosphates and from glycerol 3-phosphate to glucose 6-phosphate were determined by additions of the respective enzyme to the system. Results show that the flux control coefficients are highly modulated by the presence of allosteric effectors, as might be expected according to the regulatory properties of phosphofructokinase and fructose-1,6-bisphosphatase purified from this origin. Measured reductions of fructose 2,6-bisphosphate and AMP levels during acidosis, starvation, or after phenylephrine treatment suggest that these changes contribute to enhanced gluconeogenesis under these conditions.

The aim of this work was to study whether changes in fructose 2,6-bisphosphate concentration are correlated with variations of the glycolytic flux in the isolated working rat heart. Glycolysis was stimulated to different extents by increasing the concentration of glucose, increasing the workload, or by the addition of insulin. The glycolytic flux was measured by the rate of detritiation of [2-3H]- and [3-3H]glucose. Under all the conditions tested, an increase in fructose 2,6-bisphosphate content was observed. The glucose- or insulin-induced increase in fructose 2,6-bisphosphate content was related to an increase in the concentration of fructose 6-phosphate, the substrate of 6-phosphofructo-2-kinase. An increase in the workload correlated with a 50% decrease in the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate. Similar changes in Km have been observed when purified heart 6-phosphofructo-2-kinase was phosphorylated in vitro by the cyclic AMP-dependent protein kinase or by the calcium/calmodulin-dependent protein kinase. Since the concentration of cyclic AMP was not affected by increasing the workload, it is possible that the change in Km of 6-phosphofructo-2-kinase, which was found in hearts submitted to a high load, resulted from phosphorylation by calcium/calmodulin protein kinase; other possibilities are not excluded. Anoxia decreased the external work developed by the heart, stimulated glycolysis and glycogenolysis, but did not increase fructose 2,6-bisphosphate.

The linked equilibria involved in the binding of phosphofructokinase (EC 2.7.1.11, ATP:D-fructose-6-phosphate 1-phosphotransferase) to tubulin and microtubules were studied at high ionic strength in vitro. The concentration-dependent dissociation of phosphofructokinase was analyzed in the absence and presence of tubulin or microtubules, and the binding of kinase to the tubulin dimer and microtubules was compared. Enzyme activity of phosphofructokinase was inhibited by both tubulin and microtubules: the relative inhibition increased with decreasing enzyme concentration. The complex formation between phosphofructokinase and tubulin was demonstrated by means of fluorescent anisotropy. Concentration-dependent copelleting of the kinase with taxol-stabilized microtubules revealed binding of the enzyme to microtubules as well as phosphofructokinase-enhanced pelleting of microtubules. The binding data agree with the enzyme kinetic findings that the inactive dissociated forms of phosphofructokinase (monomer-dimer) are involved in the heterologous complex formation. Microtubule reorganization (bundle formation) by phosphofructokinase was established by turbidity measurements and sedimentation experiments. The binding data are consistent with a simple molecular model for the interactions in phosphofructokinase-tubulin/microtubules systems.

We have demonstrated previously that myocardial cytosolic calcium-independent phospholipase A2 is a 40-kDa polypeptide regulated by ligand-modulated protein-protein interactions (Hazen, S.L., and Gross, R.W. (1991) J. Biol. Chem. 266, 14526-14534). We now demonstrate that an 85-kDa polypeptide which possesses sequence homology to and chemical, physical, immunological, and chromatographic similarities with phosphofructokinase (PFK) specifically interacts with the 40-kDa phospholipase A2 catalytic subunit and represents the putative protein regulatory element identified in previous work. Multiple independent lines of evidence document the association between the 85-kDa phosphofructokinase isoform and the 40-kDa myocardial cytosolic calcium-independent phospholipase A2 catalytic polypeptide, including 1) the coelution of the 85- and 40-kDa polypeptides which migrate as a 400-kDa complex during gel filtration chromatography, 2) the stoichiometry between the 85- and 40-kDa polypeptides which corresponds to a complex comprised of a tetrameric PFK isoform and a 40-kDa phospholipase A2 catalytic polypeptide, 3) the demonstration that the 85-kDa phosphofructokinase isoform acts as a specific and reversible affinity adsorbent for myocardial cytosolic phospholipase A2 catalytic activity, 4) the immunoprecipitation of myocardial cytosolic phospholipase A2 activity utilizing chicken anti-rabbit skeletal muscle PFK IgG, 5) the specific release of phospholipase A2 from ATP-agarose after formation of a ternary complex comprised of allosteric modifiers of phosphofructokinase, and 6) the selective attenuation of the denaturation of purified homogeneous calcium-independent cytosolic phospholipase A2 with PFK. Collectively, these results demonstrate the highly specific association of a phosphofructokinase isoform with myocardial cytosolic calcium-independent phospholipase A2 and suggest a novel biochemical mechanism underlying the coordinated regulation of phospholipolysis and glycolysis previously observed in myocardium and in other mammalian tissues.

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii is a non-allosteric enzyme with properties dissimilar to those of other described phosphofructokinases. The enzyme was cloned into pBluescript, sequenced, and expressed in Escherichia coli at levels 15 times higher than those observed in Propionibacterium. The gene consists of 1215 bases which code for a protein of 404 amino acids and a mass of 43,243 daltons. High G + C in the codon usage (66%) of the gene is consistent with the classification of Propionibacterium in the High-G + C subdivision of the Gram-positive bacteria. While showing no sequence identity to the non-allosteric ATP-dependent phosphofructokinase of E. coli, alignments of the amino acid sequence with other PFKs reveal degrees of identities among the amino halves of the proteins, from 26% between the Propionibacterium and potato PPi-PFKs, and 29% between Propionibacterium and E. coli ATP-PFKs. These levels of identities indicate that the amino halves of these proteins are homologous. Identities between the carboxyl half of Propionibacterium PFK and carboxyl halves of other sequences are below 20%, suggesting that the carboxyl half is not homologous. Despite the poor conservation, most of the residues that take part in the binding of fructose-6-P or Mg-PPi could be readily identified by analogy to the structure of the E. coli PFK. Both the fructose-6-P and ATP-binding sites are conserved, indicating that PPi binds to the homologous site of the E. coli ATP-binding site.

Oscillatory behavior of glycolysis in cell-free extracts of skeletal muscle involves repeated bursts of phosphofructokinase activity and associated oscillations in the [ATP]/[ADP] ratio. Addition of citrate, a potent physiological inhibitor of phosphofructokinase, decreased the frequency of the oscillations and delayed the first burst of phosphofructokinase activity in a dose-dependent manner. Citrate decreased the trigger point [ATP]/[ADP] ratio at which bursts of phosphofructokinase activity were initiated but had a much smaller effect on the average [ATP]/[ADP] ratio and did not decrease the peak values of the ratio. When oscillations were prevented by addition of fructose-2,6-P2, the decrease in the [ATP]/[ADP] ratio caused by citrate in the steady state system was similar to the decrease in the trigger point [ATP]/[ADP] ratio in the oscillatory system. The decrease in the average [ATP]/[ADP] ratio was greater in the steady state system than in the oscillating system. These results demonstrate advantages of oscillatory behavior of glycolysis in the regulation of carbohydrate utilization and the maintenance of a high [ATP]/[ADP] ratio.

Kinetic data have been collected suggesting that heterotropic activation by fructose 2,6-bisphosphate and AMP is a result not only of the relief of allosteric inhibition by ATP but is also the result of an increase in the affinity of phosphofructokinase for fructose 6-phosphate. Modification of the Ascaris suum phosphofructokinase at the ATP inhibitory site produces a form of the enzyme that no longer has hysteretic time courses or homotropic positive (fructose 6-phosphate) cooperativity or substrate inhibition (ATP) (Rao, G.S. J., Wariso, B.A., Cook, P.F., Hofer, H.W., and Harris, B.G. (1987a) J. Biol. Chem. 262, 14068-14073). This form of phosphofructokinase is Michaelis-Menten in its kinetic behavior but is still activated by fructose 2,6-bisphosphate and AMP and by phosphorylation using the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK). Fructose 2,6-bisphosphate activates by decreasing KF-6-P by about 15-fold and has an activation constant of 92 nM, while AMP decreases KF-6-P about 6-fold and has an activation constant of 93 microM. Double activation experiments suggest that fructose 2,6-bisphosphate and AMP are synergistic in their activation. The desensitized form of the enzyme is phosphorylated by cAPK and has an increased affinity for fructose 6-phosphate in the absence of MgATP. The increased affinity results in a change in the order of addition of reactants from that with MgATP adding first for the nonphosphorylated enzyme to addition of fructose 6-phosphate first for the phosphorylated enzyme. The phosphorylated form of the enzyme is also still activated by fructose 2,6-bisphosphate and AMP.

Results of activity and spectral studies using fluorescence and circular dichroism show that AMP and fructose 2,6-bisphosphate (F-2,6-P2) activate Ascaris suum phosphofructokinase through specific and similar conformational changes. Inorganic compounds like (NH4)2SO4 and KH2PO4 also induce structural alterations in the enzyme in a manner different from those caused by AMP and F-2,6-P2. The enzyme is activated by both AMP and F-2,6-P2, in 20 mM phosphate buffer, pH 6.6, with 0.2 mM ATP and 1 mM F-6-P. The Kact values for AMP and F-2,6-P2 are 25 +/- 3 microM and 1.5 +/- 0.2 microM, respectively. Both effectors quench enzyme tryptophan fluorescence in phosphate, pH 6.6, in a concentration-dependent manner. The Kd values determined from the decrease in emission intensity at 342 nm as a function of effector concentration are 24 +/- 3 microM for AMP and 1.00 +/- 0.15 microM for F-2,6-P2, in excellent agreement with the values of Kact. Both effectors also produce dramatic changes in the CD spectrum of the enzyme, in the region from 240 to 190 nm representing the peptide backbone. Secondary structure calculations suggest an increase in the alpha-helical content of the enzyme in the presence of either effector. The Kd values obtained from the concentration dependence of the decrease in ellipticity at 210 nm are 22.8 +/- 5.3 microM and 1.3 +/- 0.2 microM, respectively, for AMP and F-2,6-P2, once again in close agreement with the Kact values for these effectors. The data imply that activation of phosphofructokinase by these effectors is concomitant with structural changes in the enzyme. Further, comparison of the difference CD spectra for the effects of AMP and F-2,6-P2 show that both of them produce similar conformational changes and probably stabilize a similar final activated state of the enzyme. Other hexose phosphate analogues such as fructose 6-phosphate, glucose 1,6-bisphosphate, and fructose 1,6-bisphosphate do not affect the CD spectrum of the enzyme. Ammonium sulfate has no effect on the CD spectrum of the enzyme in phosphate buffer but does cause a significant alteration in the spectrum obtained in Mes. Gel filtration high performance liquid chromatography using a Borosil TSK 400 column shows that the tetrameric state of the native enzyme is not affected by the presence of the effectors.

Ribose 1,5-bisphosphate (Rib-1,5-P2), a newly discovered activator of rat brain phosphofructokinase, forms rapidly during the initiation of glycolytic flux and disappears within 20 s (Ogushi, S., Lawson, J.W. R., Dobson, G.P., Veech, R.L., and Uyeda, K. (1990) J. Biol. Chem. 265, 10943-10949). Activation of various mammalian phosphofructokinases and plant pyrophosphate-dependent phosphofructokinases by Rib-1,5-P2 was investigated. The order of decreasing potency for activation of rabbit muscle phosphofructokinase was: fructose (Fru) 2,6-P2, Rib-1,5-P2, Fru-1,6-P2, Glc-1,6-P2, phosphoribosylpyrophosphate, ribulose-1,5-P2, sedoheptulose-1,7-P2, and myoinositol-1,4-P2. The K0.5 values for activation by Rib-1,5-P2 of rat brain, rat liver, and rabbit muscle phosphofructokinases and potato and mung bean pyrophosphate-dependent phosphofructokinases were 64 nM, 230 nM, 82 nM, 710 nM, and 80 microM, respectively. The corresponding K0.5 values for Fru-2,6-P2 were 9, 8.6, 10, 7, and 65 nM, respectively. Rib-1,5-P2 was a competitive inhibitor of Fru-2,6-P2, binding to the muscle enzyme with Ki of 26 microM. Citrate increased the K0.5 for Rib-1,5-P2 without affecting the maximum activation, and AMP lowered the K0.5 for Rib-1,5-P2 without affecting the maximum activation. These effects of citrate and AMP were similar to those observed with Fru-2,6-P2 and different from those with Fru-1,6-P2. Rib-1,5-P2 is the second most potent activator of phosphofructokinase thus far discovered. The Rib-1,5-P2-activated conformation of the enzyme seems to be similar to that induced by Fru-2,6-P2, but different from that induced by Fru-1,6-P2.

The tissue contents of previously known allosteric effectors of brain phosphofructokinase (EC 2.7.1.11) (PFK) and the kinetic behavior of isolated PFK were investigated during the initiation of rapid glycolytic flux in freeze-blown rat brain. Comparing 0- with 5-s brains revealed that there was a 4-fold drop in total tissue content of Fru-6-P and a 5.6-fold increase in Fru-1,6-P2 consistent with activation of PFK. Additionally, analysis of brain content showed a 15-fold increase in AMP, a 3-fold decrease in ATP, a 3-fold decrease in Pi, and a 1.6-fold increase in NH4+. There was no change in Fru-2,6-P2, H+, citrate, or Glc-1,6-P2 or the kinetic profiles of isolated PFK for ATP inhibition or Fru-2,6-P2 activation. We concluded that the observed change in PFK activity could be accounted for only partially by changes in the concentrations of adenine nucleotides and other known effectors. High performance liquid chromatography fractions of extracts obtained from 5-s brains showed the activator with a mobility identical to ribose 1,5-P2 and gave 2 nmol/g (wet weight) at 0 s, 10 nmol/g at 5 s, and 2 nmol/g at 20 s. Assay of PFK in the presence of effectors determined to be in tissue at 5 s showed that addition of 10 nmol/ml ribose 1,5-P2 gave a 4-fold activation of PFK. Based on the rapidity of its formation, its potency of activation, and its similarity in chemical properties to authentic ribose 1,5-P2, we conclude that ribose 1,5-P2 served as the initial activator of PFK in brain.

Phosphorylation of the ascarid phosphofructokinase with the catalytic subunit of beef heart cyclic AMP-dependent protein kinase results in the incorporation of 1 mol of P/mol of subunit. Accompanying the phosphorylation there is a 3-4-fold increase in catalytic activity when measured at pH 6.8 with inhibitory levels of ATP. Studies on the effect of phosphorylation on the ATP saturation curve demonstrated that phosphorylation decreased the inhibitory action of ATP. The apparent Km of the catalytic subunit for the phosphofructokinase was 11.2 microM. Chymotryptic or subtilisin digestion of the labeled enzyme released distinct but overlapping phosphopeptides that were purified by high pressure liquid chromatography and sequenced by gas phase peptide sequencing. The sequence of the chymotryptic peptide was Ala-Lys-Gly-Arg-Ser-Asp-Ser(P)-Ile-Val-Pro-Thr. Based on these results and earlier observations, it is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of energy metabolism in the parasitic helminth.

Kinetic properties of regulatory enzymes of glycolysis in liver of the mouse, Zapus hudsonius, were modified during hibernation, the probable mechanism being covalent modification. Liver glycogen phosphorylase activity was strongly depressed during both short (less than 24 h) and long (5-8 days) term hibernation, the mechanism involving a decrease in both the percentage of enzyme in the active a form and the total amount (a + b) of enzyme expressed. Phosphofructokinase showed kinetic changes (a 2.5-fold increase in Ka for fructose-2,6-P2, 4- and 3.7-fold decreases in I50 values for ATP and citrate, compared to euthermic controls) in liver of hibernators indicative of phosphorylation inactivation of the enzyme. Measured levels of fructose-2,6-P2 in liver did not change during hibernation. Changes in pyruvate kinase kinetics in liver from long term hibernators similarly indicated enzyme phosphorylation in the depressed state (Ka for fructose-1,6-P2 increased 4.4-fold, I50 for L-alanine decreased 6.3-fold). Apparent covalent modification of glycolytic enzymes during hibernation may serve two functions: depression of glycolytic activity as part of the general metabolic rate depression of hibernation, or reorganization of fuel use in the hibernating state to limit carbohydrate catabolism and promote gluconeogenesis.

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.

Linked oscillations of the glycolytic pathway and the purine nucleotide cycle were studied in particle-free extracts of rat skeletal muscle. Under the conditions used, an accumulation of about 1 muM fructose diphosphate can trigger a sudden increase in phosphofructokinase activity. The activation by fructose diphosphate depends on the presence of AMP. When the AMP concentration drops, phosphofructokinase becomes inhibited, even though the fructose disphosphate concentration remains high. It is concluded that the oscillatory behavior can be of advantage for maintaining a high average [ATP]/[ADP] ratio.

Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3-phosphoglycerate. The multienzyme reaction which produces 2 alpha-glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha-glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions.

Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was studied during differentiation of mouse C2 myoblasts to myotubes. The total PFK activity increased 20-fold during in vitro myogenesis. The rate of synthesis, relative to the rate of total protein synthesis, measured by pulse labeling and immunoprecipitation was lowest for muscle PFK (PFK-A), 0.008% in myoblasts, while those for liver (PFK-B) and brain (PFK-C) PFK were 0.017 and 0.014%, respectively. The relative rate of PFK-A synthesis increased sharply (5-fold) at an initial period of differentiation (8 h) and reached maximum of 10-fold at 48 h, to make PFK-A the major isoform synthesized in myotubes. The relative rates of synthesis for both PFK-B and PFK-C did not change drastically, decreasing slightly at 8 h, but were restored to 1.5-2-fold of myoblasts. cDNA sequences coding for mouse muscle PFK were cloned and used along with those for mouse liver PFK, which we have previously cloned, to measure by Northern blot analysis under highly stringent conditions the steady-state mRNA concentrations for muscle and liver PFK during C2 differentiation. The hybridizable mRNA level for PFK-A increased gradually, reaching 13-fold at 48 h when 80% of cells was fused to myotubes. The PFK-A mRNA level at 96 h was 90-fold of that for myoblasts. In contrast, the mRNA level for PFK-B increased slightly during differentiation, showing a maximum of 4-fold at 96 h. These results indicate isozyme-specific control of muscle PFK gene expression during C2 myoblast differentiation.

Calmodulin has been shown to interact with high affinity with muscle phosphofructokinase (Mayr, G. W. (1984) Eur. J. Biochem. 143, 513-520, 521-529). In this study, direct binding measurements indicated that each of the two subunits of dimeric phosphofructokinase bound two calmodulins with Kd values of about 3 nM and 1 microM, respectively, in a strictly Ca2+-dependent way. To get more detailed information about this interaction, calmodulin-binding fragments were isolated from a CNBr digest of phosphofructokinase using affinity chromatography on calmodulin-agarose. Two fragments, M11 (Mr 3080) and M22 (Mr 8060), formed a 1:1 stoichiometric complex with Ca2+-calmodulin. The amino acid sequences of these fragments were determined, and their positions in the three-dimensional structure-model of phosphofructokinase are proposed. Fragment M11, which binds to calmodulin with the higher affinity (Kd 11.4 nM), is located in a region of the subunit where two dimers have been proposed to make contacts if associating to active tetrameric enzyme. A stabilization of the dimeric form of the enzyme by binding of calmodulin supports this location of M11. The weaker binding fragment M22 (Kd 198 nM) corresponds to the C-terminal part of the polypeptide and contains the site which is phosphorylated by cAMP-dependent protein kinase. Both fragments have structural properties in common with the isolated calmodulin-binding domains of myosin light chain kinase: two cationic segments rich in hydrophobic residues, one constantly possessing a tryptophan, and the other exhibiting an amino acid sequence resembling sites phosphorylated by cAMP-dependent protein kinase.

The effects of insulin and increased cardiac work on glycolytic rate, metabolite content, and fructose 2,6-bisphosphate (Fru-2,6-P2) content were studied in isolated perfused rat hearts. Steady-state rates of glycolysis increased 5-fold with the addition of insulin to the perfusate or by increasing cardiac pressure-volume work and correlated well in most conditions with changes in substrate concentration (Fru-6-P) and with concentration of the activator, Fru-2,6-P2. There was no correlation with changes in other well known regulators including citrate, ATP, AMP, Pi, or cytosolic phosphorylation potential. Using phosphofructokinase purified from hearts perfused under identical conditions, allosteric kinetic experiments were performed using the metabolite and effector concentrations determined from in vivo experiments. Reaction rates for phosphofructokinase calculated in vitro agreed well with the glycolytic rates measured in vivo and correlated with changes in Fru-6-P but not with other effectors. However, higher Fru-2,6-P2 levels were more effective in maintaining phosphofructokinase activity at high ATP and citrate levels. Kinetic experiments did not indicate a covalent modification of phosphofructokinase. These data indicate that control of cardiac phosphofructokinase and glycolysis may be accomplished by changes in the availability of substrate, Fru-6-P, and activator, Fru-2,6-P2, rather than by citrate, adenine nucleotides, or cytosolic phosphorylation potential as previously suggested.

Earlier work demonstrated that the activity of liver phosphofructokinase (PFK-L2) and immunoreactive PFK-L2 were decreased in diabetic rats and increased to normal or super-normal amounts following insulin treatment (Dunaway, G.A., and Weber, G., (1974) Arch. Biochem. Biophys. 162, 629-637). This report indicates that the decrease in levels of PFK-L2 in diabetic rats is a result of an accelerated degradation rate while the synthetic rate remains nearly normal. Following insulin treatment, the rate of PFK-L2 synthesis is enhanced 2-fold, whereas the rate of degradation appears to be greatly diminished. An inverse relationship is shown to exist between the PFK-L2 levels and the rates of PFK-L2 degradation, suggesting that the levels of PFK-L2 are primarily regulated by degradation rate. In addition, the levels of the PFK-L2 peptide stabilizing factor are inversely proportional to rates of PFK-L2 degradation. These results indicate that insulin mediates the rate of degradation of PFK-L2 by controlling the level of the peptide stabilizing factor.