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Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

The mechanics of the cellular microenvironment continuously modulates cell functions such as growth, survival, apoptosis, differentiation and morphogenesis via cytoskeletal remodelling and actomyosin contractility 1 – 3 . Although all of these processes consume energy 4 , 5 , it is unknown whether and how cells adapt their metabolic activity to variable mechanical cues. Here we report that the transfer of human bronchial epithelial cells from stiff to soft substrates causes a downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by the disassembly of stress fibres, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small-cell lung cancer cells, which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress-fibre subset that is insensitive to substrate stiffness. Our data reveal a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, thus coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to tune energy production in variable microenvironments, whereas the resistance of the cytoskeleton in response to mechanical cues enables the persistence of high glycolytic rates in cancer cells despite constant alterations of the tumour tissue. Glycolysis in normal epithelial cells responds to microenvironmental mechanics via the modulation of actin bundles that sequester the phosphofructokinase-targeting ubiquitin ligase TRIM21, a process superseded by persistent actin bundles in cancer cells.

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases. Glycolytic enzymes are challenging drug targets due to their highly conserved active sites and phosphorylated substrates. Here, the authors identify fast acting allosteric inhibitors of Trypanosoma brucei phosphofructokinase that block trypanosome glycolysis and provide cure evidence in murine model.

The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (β-Lg) and alpha-lactalbumin (α-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/μg test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach.

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h−1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h−1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.

We have constructed a model of the upper part of the glycolysis in the pancreatic β-cell. The model comprises the enzymatic reactions from glucokinase to glyceraldehyde-3-phosphate dehydrogenase (GAPD). Our results show, for a substantial part of the parameter space, an oscillatory behavior of the glycolysis for a large range of glucose concentrations. We show how the occurrence of oscillations depends on glucokinase, aldolase and/or GAPD activities, and how the oscillation period depends on the phosphofructokinase activity. We propose that the ratio of glucokinase and aldolase and/or GAPD activities are adequate as characteristics of the glucose responsiveness, rather than only the glucokinase activity. We also propose that the rapid equilibrium between different oligomeric forms of phosphofructokinase may reduce the oscillation period sensitivity to phosphofructokinase activity. Methodologically, we show that a satisfying description of phosphofructokinase kinetics can be achieved using the irreversible Hill equation with allosteric modifiers. We emphasize the use of parameter ranges rather than fixed values, and the use of operationally well-defined parameters in order for this methodology to be feasible. The theoretical results presented in this study apply to the study of insulin secretion mechanisms, since glycolytic oscillations have been proposed as a cause of oscillations in the ATP/ADP ratio which is linked to insulin secretion.

Recently, unusual non-regulated ATP-dependent 6-phosphofructokinases (PFK) that belong to the PFK-B family have been described for the hyperthermophilic archaea Desulfurococcus amylolyticus and Aeropyrum pernix. Putative homologues were found in genomes of several archaea including the hyperthermophilic archaeon Methanocaldococcus jannaschii. In this organism, open reading frame MJ0406 had been annotated as a PFK-B sugar kinase. The gene encoding MJ0406 was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 68 kDa composed of 34 kDa subunits. With a temperature optimum of 85°C and a melting temperature of 90°C, the M. jannaschii nucleotide kinase represents one of the most thermoactive and thermostable members of the PFK-B family described so far. The recombinant enzyme was characterized as a functional nucleoside kinase rather than a 6-PFK. Inosine, guanosine, and cytidine were the most effective phosphoryl acceptors. Besides, adenosine, thymidine, uridin and xanthosine were less efficient. Extremely low activity was found with fructose-6-phosphate. Further, the substrate specificity of closely related PFK-Bs from D. amylolyticus and A. pernix were reanalysed.

The allosteric properties of the wild-type Escherichia coli phosphofructokinase were compared to the E187A mutant by using frequency-domain techniques. Tryptophan-shifted mutants comprising of double (W311Y/Y55W and W/311F/F188W) and triple (W311Y/Y55W/E187A and W311F/F188W/E187A) amino acid residue changes, which allowed for better fluorescence probing at targeted sites, were also compared to the wild-type and E187A. The additive nature of multiple mutations allowed one to partition the net effect of modifying residue 187. In general, the mutant enzymes displayed greater heterogeneity in sub-state population than did the wild-type enzyme. The semi-cone angle model was used to quantify the extent of depolarization of the fluorophore. Use of the model presupposes that the extent of depolarization directly correlates with the degree of flexibility of the fluorophore. A relationship has been established between the values determined from the semi-cone angle calculations and the thermodynamic components responsible for the allosteric linkage between the regulatory and substrate binding. Coupling interactions giving rise to positive entropy components are manifested by increasing flexibility of the ternary complexes rather than the binary complexes.

Abstract We have cloned a Family B sugar kinase gene from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix and have subsequently expressed the protein in Escherichia coli. The enzyme was purified with its associated histidine-tag by affinity chromatography with a nickel-nitrilotriacetic acid column followed by cation exchange chromatography and possesses a high degree of thermostable ATP-dependent phosphofructokinase activity. The enzyme has an estimated apparent Km for ATP and fructose-6-phosphate of 0.027 and 1.212 mM, respectively, that were determined in discontinuous assays at 95°C. The Family B ATP-dependent phosphofructokinase has a half-life of approximately 30 min at 95°C and is indicated to be monomeric. The implications of the presence of a Family B phosphofructokinase in the Crenarchaea are discussed with reference to the origins of the Embden–Meyerhof pathway.

Among breast cancer subtypes, the triple negative breast cancer (TNBC) has the worst prognosis. In absence of any permitted targeted therapy, standard chemotherapy is the mainstay for TNBC treatment. Hence, there is a crucial need to identify potential druggable targets in TNBCs for its effective treatment. In recent times, metabolic reprogramming has emerged as cancer cells hallmark, wherein cancer cells display discrete metabolic phenotypes to fuel cell progression and metastasis. Altered glycolysis is one such phenotype, in which even in oxygen abundance majority of cancer cells harvest considerable amount of energy through elevated glycolytic-flux. In the present review, we attempt to summarize the role of key glycolytic enzymes i.e. HK, Hexokinase; PFK, Phosphofructokinase; PKM2, Pyruvate kinase isozyme type 2; and LDH, Lactate dehydrogenase in TNBCs, and possible therapeutic options presently available.