Identification of C-terminal motifs responsible for transmission of inhibition by ATP of mammalian phosphofructokinase, and their contribution to other allosteric effects.
ABSTRACT: Systematic deletions and point mutations in the C-terminal extension of mammalian PFK (phosphofructokinase) led us to identify Leu-767 and Glu-768 of the M-type isoform (PFK-M) as the motifs responsible for the role of this region in inhibition by MgATP. These amino acids are the only residues of the C-terminus that are conserved in all mammalian isoforms, and were found to have a similar function in the C-type isoenzyme. Both residues in PFK-C and Leu-767 in PFK-M were also observed to be critical for inhibition by citrate, which is synergistic with that by MgATP. Binding studies utilizing titration of intrinsic protein fluorescence indicated that the C-terminal part of the enzyme participates in the signal transduction route from the MgATP inhibitory site to the catalytic site, but does not contribute to the binding of this inhibitor, whereas it is essential for the binding of citrate. Mutations of the identified structural motifs did not alter either the action of other allosteric effectors that also interact with MgATP, such as the inhibitor phosphoenolpyruvate and the strong activator fructose 2,6-bisphosphate, or the co-operative effect of fructose 6-phosphate. The latter data provide evidence that activation by fructose 2,6-bisphosphate and fructose 6-phosphate co-operativity are not linked to the same allosteric transition as that mediating inhibition by MgATP.
Project description:Aurintricarboxylic acid (ATA) was found to be a very potent inhibitor of purified rabbit liver phosphofructokinase (PFK), giving 50% inhibition at 0.2 microM. The inhibition was in a manner consistent with interaction at the citrate-inhibitory site of the enzyme. The data suggest that inhibition of PFK by ATA was not due to denaturation of the enzyme or the irreversible binding of inhibitor, since the inhibition could be reversed by addition of allosteric activators of PFK, i.e. fructose 2,6-bisphosphate or AMP. Two other tricarboxylic acids, agaric acid and (-)-hydroxycitrate, were found to inhibit PFK. ATA at much higher concentrations (500 microM) was shown to inhibit fatty acid synthesis from endogenous glycogen in rat hepatocytes; however, protein synthesis was not altered.
Project description:6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152?µM; K0.5F6P 147?µM), PFK-P the lowest affinities (K0.5ATP 276?µM; K0.5F6P 1333?µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160?µM; K0.5F6P 1360?µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.
Project description:The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.
Project description:Cancer cells maintain a high glycolytic rate even in the presence of oxygen, a phenomenon first described over 70 years ago and known historically as the Warburg effect. Fructose 2,6-bisphosphate is a powerful allosteric regulator of glycolysis that acts to stimulate the activity of 6-phosphofructo-1-kinase (PFK-1), the most important control point in mammalian glycolysis. The steady state concentration of fructose 2,6-bisphosphate in turn depends on the activity of the enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2, 6-bisphosphatase, which is expressed in several tissue-specific isoforms. We report herein the identification of a gene product for this enzyme that is induced by proinflammatory stimuli and which is distinguished by the presence of multiple copies of the AUUUA mRNA instability motif in its 3'-untranslated end. This inducible gene for PFK-2 is expressed constitutively in several human cancer cell lines and was found to be required for tumor cell growth in vitro and in vivo. Inhibition of inducible PFK-2 protein expression decreased the intracellular level of 5-phosphoribosyl-1-pyrophosphate, a product of the pentose phosphate pathway and an important precursor for nucleic acid biosynthesis. These studies identify a regulatory isoenzyme that may be essential for tumor growth and provide an explanation for long-standing observations concerning the apparent coupling of enhanced glycolysis and cell proliferation.
Project description: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 ?. 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.
Project description:In man and the rabbit, 6-phosphofructokinase (PFK, EC 220.127.116.11) exists in tetrameric isoenzymic forms composed of muscle (M or A), liver (L or B) and platelet or brain (P or C) subunits, which are under separate genetic control. In contrast, the genetic control of the rat PFK has not yet been conclusively established; it is unclear whether the P-type or C-type subunit exists in this species. To resolve this question, we investigated the enzyme from the skeletal muscle, liver and brain of rats of Wag/Rij strain. Our studies demonstrate that the rat PFK is also under the control of three structural loci and that the homotetramers M4, P4 and L4 exhibit unique chromatographic, immunological and kinetic-regulatory properties. Skeletal-muscle and brain PFKs consist of isolated M4 and P4 homotetramers respectively. Although liver PFK consists predominantly of L4 homotetramer, it also contains small amounts of PL3 and P2L2 species. All three PFKs exhibit allosteric properties: co-operativity with fructose 6-phosphate and inhibition by ATP decrease in the order P4 greater than L4 greater than M4. P4 and M4 tetramers are the most sensitive to citrate inhibition, whereas L4 tetramer is the least sensitive. More importantly, P4 and L4 isoenzymes are the most sensitive to activation by fructose 2,6-bisphosphate, whereas M4 isoenzyme is the least sensitive. These results indicate that the brain PFK in this strain of rat is a unique tetramer, P4, which also exhibits allosteric kinetics, as do the well-studied M4 and L4 isoenzymes. The reported differences in the number and nature of isoenzymes present in the rat brain and liver most probably reflect the differences in the strains studied by previous investigators. Since the nature of the rat PFK isoenzymes and nomenclatures reported by previous investigators have been now reconciled, it is proposed that, for the sake of uniformity, only well-established nomenclatures used for the rabbit or human PFK isoenzymes be used for the rat isoenzymes.
Project description:Cancer cells usually exhibit aberrant cell signaling and metabolic reprogramming. However, mechanisms of crosstalk between these processes remain elusive. Here, we show that in an in vivo tumor model expressing oncogenic Drosophila Homeodomain-interacting protein kinase (Hipk), tumor cells display elevated aerobic glycolysis. Mechanistically, elevated Hipk drives transcriptional upregulation of Drosophila Myc (dMyc; MYC in vertebrates) likely through convergence of multiple perturbed signaling cascades. dMyc induces robust expression of pfk2 (encoding 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase; PFKFB in vertebrates) among other glycolytic genes. Pfk2 catalyzes the synthesis of fructose-2,6-bisphosphate, which acts as a potent allosteric activator of Phosphofructokinase (Pfk) and thus stimulates glycolysis. Pfk2 and Pfk in turn are required to sustain dMyc protein accumulation post-transcriptionally, establishing a positive feedback loop. Disruption of the loop abrogates tumorous growth. Together, our study demonstrates a reciprocal stimulation of Myc and aerobic glycolysis and identifies the Pfk2-Pfk governed committed step of glycolysis as a metabolic vulnerability during tumorigenesis.
Project description:Inhibition of rat liver fructose-1,6-bisphosphatase by AMP was uncompetitive with respect to fructose 1,6-bisphosphate in the absence of fructose 2,6-bisphosphate, but non-competitive in its presence. AMP was unable to bind to the enzyme except in the presence of one of the fructose bisphosphates; the binding stoicheiometry was 2 molecules/tetramer. Increasing concentrations of Mg2+ increased the Hill coefficient h and the apparent Ki for AMP, whereas fructose 2,6-bisphosphate had the opposite effect. Increasing concentrations of both AMP and fructose 2,6-bisphosphate decreased h and increased the apparent Ka for Mg2+. AMP slightly decreased, and Mg2+ slightly increased, the apparent Ki for fructose 2,6-bisphosphate, but each had only small effects on h. These results are interpreted in terms of a new three-state model for the allosteric properties of the enzyme, in which fructose 2,6-bisphosphate can bind both to the catalytic site and to an allosteric site and AMP can bind to the enzyme only when the catalytic site is occupied.
Project description:Arthropod-borne diseases are some of the most rapidly spreading diseases. Reducing the vector population is currently the only effective way to reduce case numbers. Central metabolic pathways are potential targets to control vector populations, but have not been well explored to this aim. The information available on energy metabolism, as a way to control lifespan and dispersion through flight of dipteran vectors, is inadequate.Phosphofructokinase (PFK) activity was measured in the presence of both of its substrates, fructose-6-phosphate (F6P) and ATP, as well as some allosteric effectors: Fructose- 2,6 - bisphosphate (F2, 6BP), citrate and AMP. Aedes aegypti phosphofructokinase sequence (AaPFK) was aligned with many other insects and also vertebrate sequences. A 3D AaPFK model was produced and docking experiments were performed with AMP and citrate.The kinetic parameters of AaPFK were determined for both substrates: F6P (V?=?4.47?±?0.15 ?mol of F1, 6BP/min, K0.5?=?1.48?±?0.22 mM) and ATP (V?=?4.73?±?0.57 ?mol of F1, 6BP/min, K0.5?=?0.43?±?0.10 mM). F2,6P was a powerful activator of AaPFK, even at low ATP concentrations. AaPFK inhibition by ATP was not enhanced by citrate, consistent with observations in other insects. After examining the sequence alignment of insect and non-insect PFKs, the hypothesis is that a modification of the citrate binding site is responsible for this unique behavior. AMP, a well-known positive effector of PFK, was not capable of reverting ATP inhibition. Aedes, Anopheles and Culex are dengue, malaria and filariasis vectors, respectively, and are shown to have this distinct characteristic in phosphofructokinase control. The alignment of several insect PFKs suggested a difference in the AMP binding site and a significant change in local charges, which introduces a highly negative charge in this part of the protein, making the binding of AMP unlikely. This hypothesis was supported by 3D modeling of PFK with AMP docking, which suggested that the AMP molecule binds in a reverse orientation due to the electrostatic environment. The present findings imply a potential new way to control PFK activity and are a unique feature of these Diptera.The present findings provide the first molecular explanation for citrate insensitivity in insect PFKs, as well as demonstrating for the first time AMP insensitivity in dipterans. It also identified a potential target for novel insecticides for the control of arthropod-borne diseases.
Project description:The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na(+). Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K(+) or Cs(+). Kinetic characterization of the enzyme shows that K(+) and Na(+) alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K(+) (but not Na(+)) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K(+) (but not Na(+)) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs(+) or K(+). These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.