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Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the (βγ-bridge position of pyrophosphate to a (β-nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi. Neither back-exchange by [32p] nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction. Reduction of the pyridoxal 5'-phosphate-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 1,6-bisphosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphates probably directly coordinated to these phosphates. The pH dependence of kinetic parameters suggests that the enzyme catalyzes its reaction via general acid-base catalysis with the use of a proton shuttle. The base is required unprotonated in both reaction directions. In the direction of fructose 6-phosphate phosphorylation the base accepts a proton from the hydroxyl at C-l of F6P and then donates it to protonate the leaving phosphate. The maximum velocity of the reaction is pH independent in both reaction directions while V/K profiles exhibit pKs for binding groups (including enzyme and reactant functional groups) as well as pKs for enzyme catalytic groups. These data suggest that reactants bind only when correctly protonated and only to the correctly protonated form of the enzyme.

Phosphofructokinase (PFK) from the lactic acid bacterium Lactobacillus delbrueckii subspecies bulgaricus (LbPFK) is a non-allosteric PFK with weak binding affinity for both the allosteric ligands phospho(enol)pyruvate (PEP) and magnesium adenosine diphosphate (MgADP). PEP and MgADP bind to the same allosteric binding site but exhibit opposite effects, PEP acting as an inhibitor and MgADP an activator. In 2005, Parichatttanakul, et al. solved the first crystal structure of LbPFK to 1.87 A resolution and allowed for a structural comparison of LbPFK to the allosteric forms of PFK from E. coli (EcPFK) and Bacillus stearothermophilus (BsPFK). Two additional structures of LbPFK have been determined with the first having phosphates bound at the four active sites and four allosteric sites solved to 2.20 A resolution. The second structure solved to 1.83 A resolution contains phosphates at all eight sites with the addition of the substrate fructose-6-phosphate (F6P) in the active sites. These structures are similar to the published sulfate-bound LbPFK structure. Overall, the secondary, tertiary and quaternary structure is conserved with the exception of the residues in the allosteric site. E55, H59, S211, D214, H215 and G216, as well as the long cassettes of residues 52-61 (PFKs1) and 206-218 (PFKs2) were mutated to the corresponding residue/residues in Thermus thermophilus PFK (TtPFK). PFKs1 and PFKs1 were also combined to form PFKs1s2. The single mutations along with PFKs1 and PFKs2 showed no enhancement in PEP binding, but PFKs1s2 enhanced PEP binding 10-fold with no change in MgADP binding compared to LbPFK. D12, located along the active site interface 15 A away from the allosteric site, was mutated to an alanine and exhibited enhanced binding 9-fold for both PEP and MgADP to the allosteric binding site. A crystal structure of D12A was solved to 2.30 A resolution with sulfate bound to all eight binding sites, and showed no major changes in secondary, tertiary or quaternary structure when compared to the sulfate-bound wild-type LbPFK structure. Combining D12A with PFKs1s2 (PFKs1s2/D12A) further enhanced PEP binding with a 21-fold tighter binding compared to LbPFK with MgADP binding being similar to D12A. PEP inhibition was also quantitated in PFKs1s2/D12A with a Q_ay = 0.007 plus/minus 0.0008. Coupling between PEP and F6P in PFKs1s2D12A is 2-fold stronger than the coupling measured in EcPFK and 7-fold stronger than the coupling measured in BsPFK. The coupling measured in PFKs1s2D12A is the first measured in any of the LbPFK variants.

The preimplantation mouse embryo undergoes many metabolic changes as development proceeds. One major change is the switch from a pyruvate based metabolism, to a glucose based metabolism. The phosphofructokinase enzyme is the regulatory enzyme of glycolysis and is thought to be a major contributor in controlling the block to glycolysis in early preimplantation mouse embryos. This study was undertaken to construct a system that would allow detection of RNA for the highly glycolytically active subunit (muscletype) of the phosphofructokinase (PFK) enzyme. A muscle specific mutant PFK plasmid was generated to provide mutant internal control RNA. Using this internal control, initial reverse transcriptionpolymerase chain reaction data collected from early embryo stages suggest that the muscle type PFK subunit RNA is not expressed in the preimplantation mouse at the 1-cell or blastocyst stages. This result suggests that PFK activity detected at the later morula and blastocyst stages must be from either a different PFK subunit or a novel embryonic form of PFK. / Department of Biology

The regulatory enzyme 6-phosphofructo-l-kinase (PFK) controls the key, rate-limiting step in glycolysis. There are 3 known mammalian isoforms termed PFK-muscle (PFK-A), PFK-liver (PFK-B), and PFK-brain (PFK-C) that randomly aggregate to form active homo- and heterotetrameric isozymes with their respective frequencies and kinetic properties contingent upon the presence and concentration of individual subunits. This study utilized RT-PCR and densitometry analyses to characterize the expression patterns of the mRNA for each isoform during mouse preimplantation development. PFK-B is increasingly expressed across these stages with a significant increase in PFK-B transcript between 8-cell (0.425 ± 0.158) and morula (0.579 ± 0.197) stages (p < 0.0005). Neither PFK-A nor PFK-C mRNA was detected at any of the preimplantation stages tested. The statistically significant increase in PFK-B corresponded with the known juncture of the switch from the oxidation of maternally supplied pyruvate to a predominant glycolyticmetabolism. Such timing suggested the direct involvement of elevated PFK-B transcription with an increase in glycolysis. / Department of Biology

Sauerstoffmangel in Pflanzenzellen führt durch Hemmung der mitochondrialen Atmung zur Akkumulation von NADH und zu ATP-Mangel. Reis kann, im Gegensatz zu Weizen oder Kartoffeln, längere Sauerstoffmangel-Perioden überstehen. Ziel dieser Promotionsarbeit war es, einige Aspekte des Primär-Stoffwechsels zu untersuchen, die für das Überleben solcher Stresssituationen verantwortlich sein können, wie die Ethanol-Gärung, die Photosynthese sowie die Nutzung alternativer Energiedonoren. Die Ethanol-Gärung ermöglicht es Pflanzen, NAD zu regenerieren, und so die glycolytische ATP-Bildung aufrechtzuerhalten. Tolerante Reis-Pflanzen bildeten unter Anoxie in Dunkelheit mehr Ethanol als sensitive Weizen-Pflanzen. Dieser Unterschied war allerdings nur im Spross nachzuweisen und resultierte aus hohen Aktivitäten der Gärungsenzyme sowie aus großen Substratmengen in Reis-Blättern. Mehr als 24 h Anoxie konnten aber auch vom Reis aufgrund von Substratmangel nicht überlebt werden. Wurden Pflanzen bei Anoxie belichtet, verbesserten sich die Überlebensraten erheblich. Die Ethanol-Bildung war deutlich verringert, so dass neben der Gärung lichtabhängige Energie-liefernde Prozesse vermutet werden. Allerdings verlief die Photosynthese unter Anoxie aufgrund von CO2-Mangel nur vermindert ab. Eventuell könnte zyklischer Elektronentransport unter diesen Bedingungen zusätzliches ATP produzieren. In der Vergangenheit wurde vermutet, dass Pflanzen unter Sauerstoffmangel für Phosphorylierungsreaktionen statt ATP auch PPi nutzen könnten. In transgenen Kartoffelpflanzen, die infolge von Überexpression der E. coli-Pyrophosphatase weniger PPi enthielten als Wildtypen, wurde unter Hypoxie-Bedingungen nachgewiesen, dass PPi als alternativer Energiedonor bei der Saccharose-Spaltung eine bedeutende Rolle spielt. Dagegen konnte in transgenen Kartoffelpflanzen mit drastisch verminderter Aktivität der PPi-abhängigen Phosphofructokinase keine Beeinträchtigung der Stoffwechsel-Leistungen unter Hypoxie gezeigt werden. / Oxygen deficiency stress in plant cells leads through inhibition of mitochondrial respiration to an accumulation of NADH and a decrease in ATP content. Rice plants can survive oxygen deficiency better than wheat or potato plants. The aim of this PhD-work was to examine, which biochemical processes are responsible for plant tolerance against low oxygen stress. The studies were focused on the analysis of ethanolic fermentation, photosynthesis and the function of pyrophosphate (PPi) as an alternative energy source. By using ethanolic fermentation, plants can regenerate NAD and maintain ATP formation during glycolysis. Tolerant rice plants produced much higher amounts of ethanol during anoxia in darkness compared to sensitive wheat plants. The high fermentation rate mainly occurred in the shoots as a result of high activities of fermentative enzymes as well as high availability of carbohydrates. Nevertheless, rice plants could not survive more than 24 h of anoxia in the dark because of carbohydrate depletion. Illumination during anoxia extended survival of plants. Ethanolic fermentation rate was reduced during light exposure of plants, indicating that other energy-producing processes can compensate. However, it could be shown that the complete photosynthesis was slowed down during oxygen deficiency due to CO2 deficiency. It is likely that cyclic electron transport could at least partially contribute to ATP production during these conditions. In the past, it was speculated that PPi could replace ATP for phosphorylating processes during low oxygen stress. With transgenic potato plants expressing E. coli pyrophosphatase and therefore containing less PPi it was demonstrated that PPi is a significant alternative energy donor for sucrose cleavage during hypoxia. However, in transgenic potato plants with a reduction of synthesis and activity of PPi-dependent phosphofructokinase it could not be demonstrated that these plants suffer more from oxygen deficiency than the wildtype.

La Polyarthrite Rhumatoïde est caractérisée par une synovite à l’origine de lésions progressives ostéo-articulaires induites par les formes réactives de l’oxygène (ROS) produites par la NADPH oxydase des polynucléaires neutrophiles (PMN). La NADPH oxydase des phagocytes, est formée d’un centre catalytique membranaire, le cytochrome b558, sur lequel vient s’associer des protéines cytosoliques régulatrices (p67phox, p47phox, p40phox et Rac1/2). Nous avons étudié la spécificité de l’interaction entre la (6-phosphofructokinase 2) et de la 6PGDH (6-phosphogluconate déshydrogénase) et la NADPH oxydase des PMN. D’autre part, nous avons caractérisé les domaines de l’hétérocomplexe S100A8/A9 impliqués dans l’activation de la NADPH oxydase phagocytaire. Par ailleurs, une étude de la signature protéique dans le liquide synovial a été menée afin de rechercher l’empreinte de l’activation du PMN dans la PR.Après stimulation par le PMA, la 6PGDH et la PFK2 co-imunoprécipitent avec les facteurs cytosoliques p67phox, p47phox and p40phox. Les expériences de microscopie confocale suggèrent une co-localisation de ces deux enzymes du métabolisme du glucose avec la NADPH oxydase, dans des micro-domaines membranaires : les radeaux lipidiques. La 6PGDH est impliquée dans l’activation de la NADPH oxydase phagocytaire en élevant la concentration du NADPH cytosolique mais également en augmentant l’affinité de cette enzyme pour son substrat, le NADPH. PFK2 est l’enzyme majeure de la régulation de la glycolyse, voie est essentielle pour la production d’ATP du PMN. L’utilisation du complexe S100A8/A9 et de protéines chimères de fusion nous a permis de révéler que la partie C-terminale de S100A8 est impliquée dans la liaison avec le cytochrome b558 et l’activation de la NADPH oxydase phagocytaire. In vivo, le profil protéique du liquide articulaire de PR a révélé l’empreinte de l’activation du PMN dans cette pathologie avec une surexpression des protéines S100A8 et S100A9. Une production ectopique de S100A8/A9 par les synoviocytes de type fibroblastique a été mise en évidence.En conclusion, la 6PGDH, la PFK2 et l’hétérodimère S100A8/A9 sont de nouveaux partenaires d’activation de la NADPH oxydase des phagocytes. Dans la PR, l’activation des PMNs conduit à la sécrétion de S100A8/A9 qui semblent constituer à la fois des biomarqueurs pertinents, mais également des cibles thérapeutiques potentielles. / Rheumatoid Arthritis (RA) is caused by an inflammation of the synovial membrane leading to progressive joint destruction and deformation, related to the production NADPH oxidase related-reactive oxygen species (ROS) production. The phagocyte NADPH oxidase is a multi-protein complex formed by a catalytic core, i.e. the transmembrane cytochrome b558 and cytosolic regulators (p67phox, p47phox, p40phox and Rac1/2). We aimed at better analyzing the NADPH oxidase activation through the evaluation of the specificity of the interaction with 6PGDH or PFK2 and through the further analysis of the association with the S100A8/A9 heterocomplex. The RA-specific protein profiling was conducted in order to determine whether a PMN activation fingerprint could be revealed among RA specific proteins.Upon PMA stimulation, both 6PGDH and PFK2 co-imunoprecipitated with cytosolic factors p67phox, p47phox and p40phox. At the plasma membrane level, confocal microscopy experiments suggested a co-localization of either 6PGDH or PFK2 with the phagocyte NADPH oxidase in lipid rafts. 6PGDH enhanced the phagocyte NADPH oxidase activity by both improving the availability of cytosolic NADPH content and by increasing the affinity of the NADPH oxidase for its substrate. PFK2 also augmented the NADPH oxidase activity. PFK2 modulated the ATP concentration available for the phosphorylation of the phagocyte NADPH oxidase components and for the NDP Kinase related-Rac activation. The generation of truncated S100A8/S100A9 heterodimer chimera could reveal that the C-terminal region of S100A8 is involved in both the interaction and the activation of the phagocyte NADPH oxidase.In vivo, synovial fluid of RA patients was remarkably labelled with the PMN activation fingerprint. S100A8 and S100A9 proteins clearly distinguished RA synovial fluid from osteoarthritis and non RA-synovial fluids. An ectopic production of S100A8/S100A9 was shown in RA fibroblast like synoviocyte.In conclusion, 6PGDH, PFK2 and S100A8/A9 proteins are surrogate activating partners of the phagocyte NADPH oxidase. In RA, the activation of PMNs leads to the release of S100A8/A9 proteins which may constitute interesting biomarkers and promising therapeutic targets.

Thesis advisor: Eranthie Weerapana / The 1,3,5-triazine is a privileged scaffold in that it is planar and has three-fold symmetry which allows for controlled modification around the ring structure with various substituents. In this thesis, we report on two modular inhibitor libraries that center around a 1,3,5-triazine core scaffolding system, which have been shown to target protein disulfide isomerase A1 (PDIA1), glutaredoxin-3 (GLRX3), and 6-phosphofructo-1-kinase (PFKP). Protein disulfide isomerase A1 (PDIA1) is a thiol-disulfide oxidoreductase localized in the lumen of the endoplasmic reticulum (ER), and is an important folding catalyst and chaperone for proteins in the secretory pathway. PDIA1 contains two active-site domains (a and a’), each containing a Cys-Gly-His-Cys (CGHC) active-site motif. Here, we synthesize a targeted library o second-generation triazine-based inhibitors to optimize the potency and selectivity of our lead compound, RB-11-ca. Characterization of this targeted library afforded an optimized PDIA1 inhibitor, KSC-34, which covalently modifies C53 in the a site of PDIA1 and demonstrates time-dependent inhibition of the reductase activity of PDIA1 in vitro with a kinact/KI = 9.66 x 103 M-1s-1. Interestingly, KSC-34 treatment demonstrated that a-site inhibition led to decreased secretion of amyloidogenic antibody light chain, thus illustrating that site-selective inhibitors like KSC-34 provide useful tools for delineating the pathological role and therapeutic potential of PDIA1. In 2014, our lab first reported on RB7, a dichlorotriazine-based electrophilic small molecule which displayed extremely high reactivity and selectivity toward lysine residues in the proteome. Herein, we further on this study by investigating the unique reactivity of RB7 through the synthesis of a second-generation small molecule electrophile library and investigating proteome-wide reactivity in vitro and in situ. This library afforded KSC-46, an RB-7 analogue with p-chlorothiophenol tuning element, which provided optimal proteome reactivity to use as a scaffold for the generation of a targeted library. To take advantage of the tuned reactivity of KSC-46, a second-generation targeted library was generated to target react residues in the proteome. This library yielded two molecules, KSC-56 and KSC-65, which were identified to target glutaredoxin-3 (GLRX3) and 6-phosphofructo-1-kinase (PFKP), respectively. GLRX3 is a cytosolic, monothiol iron-sulfur cluster chaperon protein which relies on two nucleophilic cysteine residues to bind and transfer iron clusters. PFKP is known to catalyze the first irreversible step in glycolysis and regulates the flux of glucose metabolism in the cell, which makes PFKP an attract therapeutic target. KSC-56 was further characterized to bind to Cys261 in the C-terminal glutaredoxin domain of GLRX3. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The trypanosomatid parasites T. brucei, T. cruzi and Leishmania spp. are responsible for the ‘neglected diseases’ Human African Trypanosomiasis, Chagas disease and Leishmaniasis respectively. In their human infective form in the bloodstream all three trypanosomatid parasites rely heavily on glycolysis for ATP production. Phosphofructokinase (PFK) catalyses the third step of the glycolytic pathway in all organisms using aerobic respiration. It facilitates the phospho transfer from ATP to fructose 6-phosphate (F6P) to make the products fructose 1,6- bisphosphate (F16BP) and ADP. RNAi knockout of T. brucei PFK has shown the enzyme is essential for survival of the bloodstream form parasites. Trypanosomatid PFKs have a unique set of structural and regulatory differences compared to the mammalian host enzyme. These differences, coupled with the availability of trypanosomatid PFK crystal structures present an opportunity for the structure-based design of specific inhibitors against the enzyme. Here we present an enzymatic characterisation of recombinant PFKs from T. brucei, T. cruzi and Leishmania infantum trypanosomatids, their regulation by the allosteric activator AMP, and their inhibition by drug-like inhibitor compounds. Inhibitor compounds (‘CTCB compounds’) were designed against T. brucei PFK with the aim of developing novel treatments against Human African Trypanosomiasis (HAT). We describe the testing, ranking and biophysical characterisation of these compounds as part of a Wellcome Trust Seeding Drug Discovery program. We found that CTCB inhibitor compounds bound to an allosteric pocket unique to trypanosomatid PFKs. We show that the compounds are specific; neither competing with the natural substrates ATP or F6P nor inhibiting the human PFK enzyme. We describe the development and testing of highly potent and specific low molecular weight PFK inhibitors that translate to both killing of cultured T. b. brucei parasites and a cure of stage I HAT in mice models. We describe the tight, 1:1 binding of these compounds with trypanosomatid PFKs, and the thermodynamic characteristics of binding through various biophysical assays. We also show the unprecedented characterisation of the reverse PFK reaction by trypanosomatid and human forms of the enzymes. We found that PFK can also carry out the reverse enzymatic reaction, under physiologically relevant concentrations of ADP and F16BP to produce F6P and ATP. We show that the reverse reaction is also subject to allosteric regulation by AMP, and can be inhibited by the CTCB compounds with a similar potency to the forward reaction. Finally, we describe the mechanism of allosteric activation by AMP and inhibition by the drug-like compounds against trypanosomatid PFKs.

Bacillus stearothermophilus phosphofructokinase (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose-6-phosphate (F6P), binds along the other dimer-dimer interface. The different functional forms BsPFK can take when in the presence of F6P and PEP can be described by the following diproportionation equilibrium: XE + EA <--> XEA + E where XE is the enzyme bound to PEP, EA is the enzyme bound to F6P, E represents the apo enzyme, and XEA is the ternary complex formed when both substrate and inhibitor are bound. Currently in the Protein Data Bank (PDB) there are two relevant forms of wild-type BsPFK, the EA form and the X'E form, which represents the enzyme bound to the PEP analog, phosphoglycolate (PGA). When comparing the EA and the X'E structures, a 7° rotation about the substrate-binding interface is observed and is termed the quaternary shift. The current study uses methyl TROSY NMR to examine the different liganded states of BsPFK, and for the first time structural data for the XEA species is shown. In addition, crystallography was used to obtain the first apo structure of BsPFK. To distinguish between changes associated with the quaternary shift and those associated with the intra-subunit tertiary changes, the variant D12A BsPFK was studied using kinetics, crystallography, and NMR. Crystal structures of apo and PEP bound forms of D12A BsPFK both indicate a shifted structure similar to the X'E form of wild-type. Kinetic studies of D12A BsPFK, when compared to wild-type, show a 50-fold diminished F6P binding affinity, 100-fold enhanced binding affinity, and a similar coupling constant. A conserved hydrogen bond between D12 and T156 takes place across the substrate binding interface in the EA form of BsPFK. The variant T156A BsPFK shows similar binding, coupling, and structural characteristics to D12A BsPFK. PEP still inhibits these variants of BsPFK despite the fact that the enzymes are in the quaternary shifted position prior to PEP binding. Therefore the quaternary shift of BsPFK primarily perturbs ligand binding but does not directly contribute to heterotropic allosteric inhibition.