Structures of human phosphofructokinase-1 and atomic basis of cancer-associated mutations.

Phosphofructokinase-1 (PFK1), the 'gatekeeper' of glycolysis, catalyses the committed step of the glycolytic pathway by converting fructose-6-phosphate to fructose-1,6-bisphosphate. Allosteric activation and inhibition of PFK1 by over ten metabolites and in response to hormonal signalling fine-tune glycolytic flux to meet energy requirements. Mutations inhibiting PFK1 activity cause glycogen storage disease type VII, also known as Tarui disease, and mice deficient in muscle PFK1 have decreased fat stores. Additionally, PFK1 is proposed to have important roles in metabolic reprogramming in cancer. Despite its critical role in glucose flux, the biologically relevant crystal structure of the mammalian PFK1 tetramer has not been determined. Here we report the first structures of the mammalian PFK1 tetramer, for the human platelet isoform (PFKP), in complex with ATP-Mg(2+) and ADP at 3.1 and 3.4 Å, respectively. The structures reveal substantial conformational changes in the enzyme upon nucleotide hydrolysis as well as a unique tetramer interface. Mutations of residues in this interface can affect tetramer formation, enzyme catalysis and regulation, indicating the functional importance of the tetramer. With altered glycolytic flux being a hallmark of cancers, these new structures allow a molecular understanding of the functional consequences of somatic PFK1 mutations identified in human cancers. We characterize three of these mutations and show they have distinct effects on allosteric regulation of PFKP activity and lactate production. The PFKP structural blueprint for somatic mutations as well as the catalytic site can guide therapeutic targeting of PFK1 activity to control dysregulated glycolysis in disease.

Structural Insights into the Regulation of Staphylococcus aureus Phosphofructokinase by Tetramer-Dimer Conversion.

Most reported bacterial phosphofructokinases (Pfks) are tetramers that exhibit activity allosterically regulated via conformational changes between the R and T states. We report that the Pfk from Staphylococcus aureus NCTC 8325 ( SaPfk) exists as both an active tetramer and an inactive dimer in solution. Multiple effectors, including pH, ADP, ATP, and adenylyl-imidodiphosphate (AMP-PNP), cause equilibrium shifts from the tetramer to dimer, whereas the substrate F6P stabilizes SaPfk tetrameric assembly. Crystal structures of SaPfk in complex with different ligands and biochemical analysis reveal that the flexibility of the Gly150-Leu151 motif in helix α7 plays a role in tetramer-dimer conversion. Thus, we propose a molecular mechanism for allosteric regulation of bacterial Pfk via conversion between the tetramer and dimer in addition to the well-characterized R-state/T-state mechanism.

Crystal structure of human platelet phosphofructokinase-1 locked in an activated conformation.

Phosphofructokinase-1 (Pfk) acts as the main control point of flux through glycolysis. It is involved in complex allosteric regulation and Pfk mutations have been linked to cancer development. Whereas the 3D structure and structural basis of allosteric regulation of prokaryotic Pfk has been studied in great detail, our knowledge about the molecular basis of the allosteric behaviour of the more complex mammalian Pfk is still very limited. To characterize the structural basis of allosteric regulation, the subunit interfaces and the functional consequences of modifications in Tarui's disease and cancer, we analysed the physiological homotetramer of human platelet Pfk at up to 2.67 Å resolution in two crystal forms. The crystallized enzyme is permanently activated by a deletion of the 22 C-terminal residues. Complex structures with ADP and fructose-6-phosphate (F6P) and with ATP suggest a role of three aspartates in the deprotonation of the OH-nucleophile of F6P and in the co-ordination of the catalytic magnesium ion. Changes at the dimer interface, including an asymmetry observed in both crystal forms, are the primary mechanism of allosteric regulation of Pfk by influencing the F6P-binding site. Whereas the nature of this conformational switch appears to be largely conserved in bacterial, yeast and mammalian Pfk, initiation of these changes differs significantly in eukaryotic Pfk.

Characterization of the pyrophosphate-dependent 6-phosphofructokinase from Xanthomonas campestris pv. campestris.

Xanthomonads are plant pathogenic proteobacteria that produce the polysaccharide xanthan. They are assumed to catabolize glucose mainly via the Entner-Doudoroff pathway. Whereas previous studies have demonstrated no phosphofructokinase (PFK) activity in xanthomonads, detailed genome analysis revealed in Xanthomonas campestris pathovar campestris (Xcc) genes for all Embden-Meyerhof-Parnas pathway (glycolysis) enzymes, including a conserved pfkA gene similar to 6-phosphofructokinase genes. To address this discrepancy between genetic and physiological properties, the pfkA gene of Xcc strain B100 was cloned into the expression vector pET28a+. The 45-kDa pfkA gene product exhibited no conventional PFK activity. Bioinformatic analysis of the Xcc PfkA amino acid sequence suggested utilization of pyrophosphate as an alternative cosubstrate. Pyrophosphate-dependent PFK activity was shown in an in vitro enzyme assay for purified Xcc PfkA, as well as in the Xcc B100 crude protein extract. Kinetic constants were determined for the forward and reverse reactions. Primary structure conservation indicates the global presence of similar enzymes among Xanthomonadaceae.

Herpes simplex type 1 activates glycolysis through engagement of the enzyme 6-phosphofructo-1-kinase (PFK-1).

UNLABELLED: Viruses such as HIV, HCV, Mayaro and HCMV affect cellular metabolic pathways, including glycolysis. Although some studies have suggested that the inhibition of glycolysis affects HSV-1 replication and that HSV-1-infected eyes have increased lactate production, the mechanisms by which HSV-1 induces glycolysis have never been investigated in detail. In this study, we observed an increase in glucose uptake, lactate efflux and ATP content in HSV-1-infected cells. HSV-1 triggered a MOI-dependent increase in the activity of phosphofructokinase-1 (PFK-1), a key rate-limiting enzyme of the glycolytic pathway. After HSV-1 infection, we observed increased PFK-1 expression, which increased PFK-1 total activity, and the phosphorylation of this enzyme at serine residues. HSV-1-induced glycolysis was associated with increased ATP content, and these events were critical for viral replication. In summary, our results suggest that HSV-1 triggers glycolysis through a different mechanism than other herpesviruses, such as HCMV. Thus, this study contributes to a better understanding of HSV-1 pathogenesis and provides insights into novel targets for antiviral therapy. HIGHLIGHTS: ►HSV-1 activates glycolysis by PFK-1 activation. ►In HSV-1-infected cells PFK-1 synthesis is up-regulated and phosphorylated at serine residues. ►PFK-1 knockdown impairs HSV-1 replication. ►HSV-1-mediated glycolysis activation increases ATP content.

Enzymes of inorganic polyphosphate metabolism.

Inorganic polyphosphate (PolyP) is a linear polymer containing a few to several hundred orthophosphate residues linked by energy-rich phosphoanhydride bonds. Investigation of PolyP-metabolizing enzymes is important for medicine, because PolyPs perform numerous functions in the cells. In human organism, PolyPs are involved in the regulation of Ca(2+) uptake in mitochondria, bone tissue development, and blood coagulation. The essentiality of polyphosphate kinases in the virulence of pathogenic bacteria is a basis for the discovery of new antibiotics. The properties of the major enzymes of PolyP metabolism, first of all polyphosphate kinases and exopolyphosphatases, are described in the review. The main differences between the enzymes of PolyP biosynthesis and utilization of prokaryotic and eukaryotic cells, as well as the multiple functions of some enzymes of PolyP metabolism, are considered.

Structure and allosteric regulation of eukaryotic 6-phosphofructokinases.

Although the crystal structures of prokaryotic 6-phosphofructokinase, a key enzyme of glycolysis, have been available for almost 25 years now, structural information about the more complex and highly regulated eukaryotic enzymes is still lacking until now. This review provides an overview of the current knowledge of eukaryotic 6-phosphofructokinase based on recent crystal structures, kinetic analyses and site-directed mutagenesis data with special focus on the molecular architecture and the structural basis of allosteric regulation.

The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition.

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.

Altered allosteric regulation of muscle 6-phosphofructokinase causes Tarui disease.

Tarui disease is a glycogen storage disease (GSD VII) and characterized by exercise intolerance with muscle weakness and cramping, mild myopathy, myoglobinuria and compensated hemolysis. It is caused by mutations in the muscle 6-phosphofructokinase (Pfk). Pfk is an oligomeric, allosteric enzyme which catalyzes one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk displayed several allosteric adenine nucleotide binding sites. Functional studies revealed a reciprocal linkage between the activating and inhibitory allosteric binding sites. Herein, we showed that Asp(543)Ala, a naturally occurring disease-causing mutation in the activating binding site, causes an increased efficacy of ATP at the inhibitory allosteric binding site. The reciprocal linkage between the activating and inhibitory binding sites leads to reduced enzyme activity and therefore to the clinical phenotype. Pharmacological blockage of the inhibitory allosteric binding site or highly efficient ligands for the activating allosteric binding site may be of therapeutic relevance for patients with Tarui disease.

Molecular architecture and structural basis of allosteric regulation of eukaryotic phosphofructokinases.

Eukaryotic ATP-dependent 6-phosphofructokinases (Pfks) differ from their bacterial counterparts in a much more complex structural organization and allosteric regulation. Pichia pastoris Pfk (PpPfk) is, with ∼ 1 MDa, the most complex and probably largest eukaryotic Pfk. We have determined the crystal structure of full-length PpPfk to 3.05 Å resolution in the T state. PpPfk forms a (αßγ)(4) dodecamer of D(2) symmetry with dimensions of 161 × 157 × 233 Å mainly via interactions of the α chains. The N-terminal domains of the α and ß chains have folds that are distantly related to glyoxalase I, but the active sites are no longer functional. Interestingly, these domains located at the 2 distal ends of this protein along the long 2-fold axis form a (αß)(2) dimer as does the core Pfk domains; however, the domains are swapped across the tetramerization interface. In PpPfk, the unique γ subunit participates in oligomerization of the αß chains. This modulator protein was acquired from an ancient S-adenosylmethionine-dependent methyltransferase. The identification of novel ATP binding sites, which do not correspond to the bacterial catalytic or effector binding sites, point to marked structural and functional differences between bacterial and eukaryotic Pfks.

Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060.

The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His(6)-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.

Potential allosteric sites captured in glycolytic enzymes via residue-based network models: Phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase.

Likelihood of new allosteric sites for glycolytic enzymes, phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GADPH) and pyruvate kinase (PK) was evaluated for bacterial, parasitic and human species. Allosteric effect of a ligand binding at a site was revealed on the basis of low-frequency normal modes via Cα-harmonic residue network model. In bacterial PFK, perturbation of the proposed allosteric site outperformed the known allosteric one, producing a high amount of stabilization or reduced dynamics, on all catalytic regions. Another proposed allosteric spot at the dimer interface in parasitic PFK exhibited major stabilization effect on catalytic regions. In parasitic GADPH, the most desired allosteric response was observed upon perturbation of its tunnel region which incorporated key residues for functional regulation. Proposed allosteric site in bacterial PK produced a satisfactory allosteric response on all catalytic regions, whereas in human and parasitic PKs, a partial inhibition was observed. Residue network model based solely on contact topology identified the 'hub residues' with high betweenness tracing plausible allosteric communication pathways between distant functional sites. For both bacterial PFK and PK, proposed sites accommodated hub residues twice as much as the known allosteric site. Tunnel region in parasitic GADPH with the strongest allosteric effect among species, incorporated the highest number of hub residues. These results clearly suggest a one-to-one correspondence between the degree of allosteric effect and the number of hub residues in that perturbation site, which increases the likelihood of its allosteric nature.

Muscle-type 6-phosphofructo-1-kinase and aldolase associate conferring catalytic advantages for both enzymes.

6-Phosphofructo-1-kinase (PFK) and aldolase are two sequential glycolytic enzymes that associate forming heterotetramers containing a dimer of each enzyme. Although free PFK dimers present a negligible activity, once associated to aldolase these dimers are as active as the fully active tetrameric conformation of the enzyme. Here we show that aldolase-associated PFK dimers are not inhibited by clotrimazole, an antifungal azole derivative proposed as an antineoplastic drug due to its inhibitory effects on PFK. In the presence of aldolase, PFK is not modulated by its allosteric activators, ADP and fructose-2,6-bisphosphate, but is still inhibited by citrate and lactate. The association between the two enzymes also results on the twofold stimulation of aldolase maximal velocity and affinity for its substrate. These results suggest that the association between PFK and aldolase confers catalytic advantage for both enzymes and may contribute to the channeling of the glycolytic metabolism.

Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition in phosphofructokinase from Escherichia coli.

This study quantifies the contribution of each of the four unique inhibiting heterotropic interactions between the allosteric inhibitor, phosphoenolpyruvate (PEP), and the substrate, fructose 6-phosphate (Fru-6-P), in phosphofructokinase from Escherichia coli (EcPFK). The unique heterotropic interactions, previously labeled by the distances between ligand binding sites, were isolated independently by constructing hybrid tetramers. Of the four unique heterotropic PEP-Fru-6-P interactions, the 45 A interaction contributed 25%, the 30 A interaction contributed 31%, and the 23 A interaction contributed 42% of the total PEP inhibition. The 33 A interaction actually causes a small activation of Fru-6-P binding by PEP and therefore contributed -8% of the total observed PEP inhibition. The pattern of relative contribution to PEP inhibition from each interaction in EcPFK does not follow the same pattern seen in MgADP activation of EcPFK. This observation supports the conclusion that although PEP and MgADP bind to the same site, they do not use the same communication pathways to influence the active site. The pattern of relative contribution describing PEP inhibition observed in this study also does not follow the pattern determined for PEP inhibition in phosphofructokinase from Bacillus stearothermophilus, suggesting that these two highly homologous isoforms are not inhibited in the same manner by PEP.

Diacylglyceride kinases, sphingosine kinases and NAD kinases: distant relatives of 6-phosphofructokinases.

Diacylglyceride kinases, sphingosine kinases, NAD kinases and 6-phosphofructokinases are thought to be related despite large evolution of their sequences. Discovery of a common signature has led to the suggestion that they possess a similar phosphate-donor-binding site and a similar phosphorylation mechanism. The substrate- and allosteric-binding sites are much more divergent and their delineation remains to be determined experimentally.

Toward Comprehensive Allosteric Control over Protein Activity.

Universality of allosteric signaling in proteins, molecular machines, and receptors complemented by the great advantages of prospected allosteric drugs in the highly specific, non-competitive, and modulatory nature of their actions calls for deeper theoretical understanding of allosteric communication. We present a computational model that makes it possible to tackle the problem of modulating the energetics of protein allosteric communication. In the context of the energy landscape paradigm, allosteric signaling is always a result of perturbations, such as ligand binding, mutations, and intermolecular interactions. The calculation of local partition functions in the protein harmonic model with perturbations allows us to evaluate the energetics of allosteric communication at the single-residue level. In this framework, Allosteric Signaling Maps are proposed as a tool to exhaustively describe allosteric communication in the protein, to tune already existing signaling, and to design new elements of regulation for taking the protein activity under allosteric control.

Phosphofructokinase-1 and fructose bisphosphatase-1 in canine liver and kidney.

In healthy individuals, plasma glucose levels are maintained within a normal range. During fasting, endogenous glucose is released either through glycogenolysis or gluconeogenesis. Gluconeogenesis involves the formation of glucose-6-phosphate from a variety of precursors followed by its subsequent hydrolysis to glucose. Gluconeogenesis occurs in the liver and the kidney. In order to compare gluconeogenesis in canine liver and kidney, the activity and expression of the rate limiting enzymes that catalyze the fructose-6-phosphate and fructose 1,6-bisphosphate steps, namely, phosphofructokinase-1 (PFK-1) (glycolysis) and fructose bisphosphatase-1 (FBP-1) (gluconeogenesis), were examined. Healthy male and female beagle dogs aged 1-2 years were euthanized humanely, and samples of their liver and kidney were obtained for analysis. The levels of PFK-1 and FBP-1 in canine liver and kidney were assessed by enzymatic assays, Western blotting, and RT-qPCR. Enzyme assays showed that, in dogs, the kidney had higher specific activity of PFK-1 and FBP-1 than the liver. Western blotting and RT-qPCR data demonstrated that of the three different subunits (PFK-M, PFK-L, and PFK-P) the PFK-1 in canine liver mainly comprised PFK-L, whereas the PFK-1 in the canine kidney comprised all three subunits. As a result of these differences in the subunit composition of PFK-1, glucose metabolism might be regulated differently in the liver and kidney.

Phosphofructokinase-1 subunit composition and activity in the skeletal muscle, liver, and brain of dogs.

Phosphofructokinase-1 (EC:2.7.1.11, PFK-1) catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate using adenosine triphosphate and is a key regulatory enzyme of glycolysis. Mammalian PFK-1 isozymes are composed of three kinds of subunits (PFK-M, -L, and -P), with different properties. It has been suggested that the proportion of PFK-1 subunits in different organs is based on the organ energy metabolism. In this study, we analyzed the activity and subunit composition of canine PFK-1. We found that, in dogs, the skeletal muscle only has PFK-M, the liver mainly has PFK-L, and the brain expresses all of them. The knowledge of the composition of PFK-1 could provide useful information for determination of the differences in glycolysis in various organs of dogs.

Purification and kinetic properties of 6-phosphofructo-1-kinase from gilthead sea bream muscle.

The kinetic properties of 6-phosphofructo-1-kinase (PFK) from skeletal muscle (PFKM) of gilthead sea bream (Sparus aurata) were studied, after 10,900-fold purification to homogeneity. The native enzyme had an apparent molecular mass of 662 kDa and is composed of 81 kDa subunits, suggesting a homooctameric structure. At physiological pH, S. aurata PFKM exhibited sigmoidal kinetics for the substrates, fructose-6-phosphate (fru-6-P) and ATP. Fructose-2,6-bisphosphate (fru-2,6-P(2)) converted the saturation curves for fru-6-P to hyperbolic, activated PFKM synergistically with other positive effectors of the enzyme such as AMP and ADP, and counteracted ATP and citrate inhibition. The fish enzyme showed differences regarding other animal PFKs: it is active as a homooctamer, and fru-2,6-P(2) and pH affected affinity for ATP. By monitoring incorporation of (32)P from ATP, we show that fish PFKM is a substrate for the cAMP-dependent protein kinase. The mechanism involved in PFKM activation by phosphorylation contrasts with previous observations in other species: it increased V(max) and did not affect affinity for fru-6-P. Unlike the mammalian muscle enzyme, our findings support that phosphorylation of PFKM may exert a major role during starvation in fish muscle.

Kinetic model of phosphofructokinase-1 from Escherichia coli.

This paper presents a kinetic model of phosphofructokinase-1 from Escherichia coli. A complete catalytic cycle has been reconstructed based on available information on the oligomeric structure of the enzyme and kinetic mechanism of its monomer. Applying the generalization of the Monod-Wyman-Changeux approach proposed by Popova and Sel'kov(35-37) to the reconstructed catalytic cycle rate equation has been derived. Dependence of the reaction rate on pH, magnesium, and effectors has been taken into account. Kinetic parameters have been estimated via fitting the rate equation against experimentally measured dependencies of initial rate on substrates, products, effectors, and pH available from the literature. The model of phosphofructokinase-1 predicts (1) cooperativity of binding both fructose-6-phosphate and ATPMg(2-), (2) significant inhibition of the enzyme resulting from an increase in total concentration of ATP under the condition of fixed concentration of Mg(2+) ions, and (3) dual effect of ADP consisting of allosteric activation and product inhibition of the enzyme. Moreover, the model developed can be used in the kinetic modeling of biochemical pathways containing phosphofructokinase-1.