Dissertations / Theses on the topic 'Glycolytic enzymes'
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1
Crowhurst, Georgina Sheila Ellen. "Studies with hyperthermophilic archaeal glycolytic enzymes." Thesis, University of Exeter, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324719.
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Duncan, John Andrew Carleton University Dissertation Biology. "Glycolytic enzyme binding and metabolic control." Ottawa, 1988.
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Yuan, Meng. "A study of regulatory mechanisms of glycolytic and gluconeogenic enzymes." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25725.
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Many diseases correlate with abnormal glucose metabolism in cells and organisms. For instance, the human M2 isoform of the glycolytic enzyme pyruvate kinase (M2PYK) plays an important role in metabolic reprogramming of tumour cells whereby aerobic glycolysis or the ‘Warburg effect’ supports cell proliferation by accumulating necessary biomass. By contrast, gluconeogenesis may play an important role, as observed in certain types of trypanosomatid parasites (e.g. the amastigote form of Leishmania major) where anabolism is essential for infectious properties. Hence, these glucose metabolising enzymes are important potential drug targets for cancer and trypanosomiasis. However, many aspects of their regulatory mechanisms are still poorly understood. This thesis describes biochemical and structural studies on M2PYK and on L. major fructose-1,6-bisphosphatase (LmFBPase), providing insights into allosteric mechanisms and structure-based drug design for both enzymes. Human PYKs and LmFBPase were expressed and purified from Escherichia coli, and their kinetics were fully characterised. It was shown that certain amino acids regulate the activity of M2PYK allosterically, but in opposite ways, with some being inhibitors and others activators. X-ray crystallographic structures and biophysical data of M2PYK complexes with alanine, phenylalanine, serine or tryptophan reveal an R-/T-state oscillating model of M2PYK involving a 11° rotation of each subunit. In addition, M2PYK was demonstrated to be a redox-sensitive enzyme. Reducing reagents were shown to help maintain the tetramer and prevent its dissociation, and thereby to activate M2PYK, whereas oxidation and nitrosylation reagents functioned in the opposite sense. Nitrosylation assays showed that the main nitrosylated residue is Cys326 of M2PYK, which is located on the tetramer interface. Dynamics and modulator effects of PYKs were further studied by hydrogen–deuterium exchange by mass spectrometry. These observations highlight the important effects of amino acids on M2PYK regulation. M1PYK by contrast, was demonstrated to be a constitutively fully active pyruvate kinase, with minor effects from modulators. The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is a potential drug target against leishmaniasis. Here we present biochemical and structural studies for LmFBPase, by characterising its activity in a metal-dependent reaction, as well as its inhibition by AMP. The crystal structure of LmFBPase is a homotetramer, composed of monomers with alternating α/β/α/β/α ‘club sandwich’ topologies. In comparison with previously revealed LmFBPase structures, the AMP-complexed structure shows a rotated form of the tetramer. Comparisons of the structures reveal an ‘unlock-androtate’ allosteric mechanism in which AMP binding causes a series of structural changes culminating in an incomplete and non-productive active site. The structure of the effector site of LmFBPase shows a different conformation from human FBPases, thereby offering a potential specific target for Leishmania.
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Bawa, Simranjot. "Exploring the molecular mechanisms of Drosophila dTRIM32 implicated in pathogenesis of Limb-Girdle Muscular Dystrophy 2H." Thesis, Kansas State University, 2017. http://hdl.handle.net/2097/38243.
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Master of ScienceBiochemistry and Molecular Biophysics Interdepartmental Program
Erika Rae Geisbrecht
The E3 ubiquitin ligase TRIM32 is a member of tripartite motif (TRIM) family of proteins involved in various processes including differentiation, cell growth, muscle regeneration and cancer. TRIM32 is conserved between vertebrates (humans, mouse) and invertebrates (Drosophila). The N-terminus of this protein is characterized by a RING domain, B-box domain, and Coiled-Coil region, while the C-terminus contains six NHL repeats. In humans, mutations that cluster in the NHL domains of TRIM32 result in the muscle disorders Limb-Girdle Muscular Dystrophy type 2H (LGMD2H) and Sarcotubular Myopathy (STM). Mutations in the B-box region cause Bardet-Biedl Syndrome (BBS), a clinically separate disorder that affects multiple parts of the body. A comprehensive genetic analysis in vertebrate models is complicated by the ubiquitous expression of TRIM32 and neurogenic defects in TRIM32-/- mutant mice that are independent of the muscle pathology associated with LGMD2H. The model organism Drosophila melanogaster possesses a TRIM32 [dTRIM32/Thin (Tn)/Abba] homolog highly expressed in muscle tissue. We previously showed that dTRIM32 is localized to Z-disk of the sarcomere and is required for myofibril stability. Muscles form correctly in Drosophila tn mutants, but exhibit a degenerative muscle phenotype once contraction ensues. Mutant or RNAi knockdown larvae are also defective in locomotion, which mimics clinical features associated with loss of TRIM32 in LGMD2H patients. It is predicted that mutations in the NHL domain either affect protein structure or are involved in protein-protein interactions. However, the molecular mechanism by which these mutations affect the interaction properties of dTRIM32 is not understood. Biochemical pulldown assays using the bait fusion protein GST-dTRIM32-NHL identified numerous dTRIM32 binding proteins in larval muscle tissue. Many key glycolytic enzymes were present in the dTRIM32 pulldowns and not in control experiments. Glycolytic genes are expressed in the developing Drosophila musculature and are required for myoblast fusion. Strikingly, many glycolytic proteins are also found at the Z-disk, consistent with dTRIM32 localization. Our biochemical and genetic studies provide evidence that there is direct interaction between dTRIM32 and glycolytic proteins (Aldolase and PGLYM). dTRIM32 also regulates glycolytic enzyme levels and protein localization at their sites of action. These data together suggest a role for dTRIM32 in coordinating glycolytic enzyme function, possibly for localized ATP production or to maintain muscle mass via glycolytic intermediates.
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Shanmuganathan, Anupama. "An Analysis of Glycolytic Enzymes in the Cellular Response to Metal Toxicity." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/biology_diss/63.
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Metal toxicity is implicated in neurotoxicity, nephrotoxicity, aging and cancer. Protein oxidation resulting from oxidative stress is now known to be involved in metal toxicity. However, proteomic responses to metal induced oxidative stress have not been characterized. By using the yeast as a model, we characterized these changes occurring in response to sub-lethal doses of metals. Several proteins involved in protein synthesis, ribosome assembly decreased while antioxidant defenses, proteins involved in sulfur metabolism, and glutathione synthesis and ubiquitin increased following metal exposure. We also show that metals induced temporal and targeted protein oxidation independent of protein abundance. Among the targets were glycolytic enzymes and heat-shock proteins. As a consequence, glycolytic enzyme activities decreased whereas the levels and activities of the enzymes of the alternative pathway for glucose metabolism, pentose phosphate pathway (PPP) increased. True to prediction, we also found increased flow through the PPP as measured by elevated levels of NADPH and glutathione. NADPH and glutathione are crucial for maintaining the redox balance in the cell. Thus, rerouting of glucose metabolism into PPP is considered to be beneficial to the organism. Among the oxidation targets is a glycolytic protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that is required for apoptosis in neuronal cells. We show that not only is GAPDH required for metal induced apoptosis in yeast but also the levels of GAPDH transcript and protein increase in the cytosol and the nucleus in an isoform specific fashion. Such changes strongly implicate the role of GAPDH in yeast apoptosis. This work provides evidence for the involvement of targeted protein oxidation in metal toxicity, shows the overlaps and differences in the mechanism of copper and cadmium toxicity, allows comprehension of how metabolic processes respond to metal stress and explores the potential of GAPDH as a sensor of oxidative stress and mediator for apoptosis.
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Abdulla, Sheera. "Biochemical characterisation of unusual glycolytic enzymes from the human intestinal parasite Blastocystis hominis." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/23933.
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Blastocystis is an important parasite that infects humans and a wide range of animals like rats, birds, reptiles, etc. infecting a sum of 60% of world population. It belongs to the Stramenopiles, a Heterologous group that includes for example the Phythophthora infestans the responsible for the Irish potato famine. Previous work had reported the presence of an unusual fusion protein that is composed of two of the main glycolytic enzymes; Triosephosphate isomerase-glyceraldehyde-3-phosphate dehydrogenase (TPI-GAPDH). Little is known about this protein. Blastocystis TPI-GAPDH and Blastocystis enolase were both characterized biochemically and biophysically in this project. The phylogenetic relationships of those two proteins among other members of either Stramenopiles, or other members of the kingdom of life were examined and found to be grouping within the chromalveolates. Our studies revealed that those two proteins, Blastocystis enolase and Blastocystis TPI-GAPDH, had a peptide signal targeting them to the mitochondria. This was an unusual finding knowing that text books always referred to the glycolytic pathway as a canonical cytoplasmic pathway. Structural studies had also been conducted to unravel the unknown structure of the fusion protein Blastocystis TPI-GAPDH. X-ray crystallography had been conducted to solve the protein structure and the protein was found to be a tetrameric protein composed of a central tetrameric GAPDH protein flanked with two dimmers of TPI protein. Solving its structure would be the starting point towards reviling the role that TPI-GAPDH might play in Blastocystis and other organisms that it was found in as well. Although a fusion protein, the individual components of the fusion were found to contain all features deemed essential for function for TPI and GAPDH and contain all expected protein motifs for these enzymes.
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Peshavaria, Mina. "Structure and regulation of the human muscle-specific enolase gene." Thesis, University of Southampton, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295627.
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Pearce, Amanda K. "Regulation of glycolysis in Saccharomyces cerevisiae." Thesis, University of Aberdeen, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301297.
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This thesis extends the work of Crimmins (1995) on the control of glycolytic flux in yeast by the enzymes 6-phosphofructo-1-kinase and pyruvate kinase (Pyk1p). This study also examines the influence of Pf1kp and Pyk1p upon yeast resistance to the weak acid preservative, benzoic acid. In Saccharomyces cerevisiae, Pyk1p is encoded by PYK1, and the α and β subunits of Pf1kp are encoded by PFK1 and PFK2, respectively. To test the influence of these genes upon glycolytic control, an isogenic set of S. cerevisiae mutants were utilised in which PYK1, PFK1 and PFK2 expression is dependent on the PGK1 promoter. Increased Pf1k levels had little effect upon rates of glucose utilisation or ethanol production during fermentative growth. However, overexpressing Pyk1p resulted in an increased growth rate and an increase in glycolytic flux. This suggests that Pyk1p, but not Pf1kp, exerts some degree of control over the glycolytic flux under these conditions. The effects of reducing Pf1kp and Pyk1p levels were also studied by placing PYK1, PFK1 and PFK2 under the control of the weak PGK1Δuas promoter. The double Pf1kp mutant showed no significant changes in doubling time, ethanol production or glucose consumption. However, a mutant with a 3-fold reduction ion Pyk1p levels displayed slower growth rates and reduced glycolytic flux. In addition, there was an imbalance in the carbon flow in this mutant, with reductions in ethanol and glycerol production evident, along with increased TCA cycle activity. Hence, while Pf1kp levels did not affect cell physiology significantly under the conditions studied, reduced Pyk1p levels seemed to disturb glycolytic flux and carbon flow. Decreased Pf1kp levels caused an increase in the sensitivity of yeast cells to benzoate, whereas the Pyk1p mutant was not affected. This confirmed that benzoic acid specifically inhibits Pf1kp rather than glycolysis in general.
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Xintaropoulou, Chrysi. "Targeting aerobic glycolysis in breast and ovarian cancer." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/29525.
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Cancer cells, unlike normal tissue, frequently rely on glycolysis for the production of energy and the metabolic intermediates required for their growth regardless of cellular oxygenation levels. This metabolic reconfiguration, termed the Warburg effect, provides a potential strategy to preferentially target tumours from a therapeutic perspective. The present study sought to investigate the glycolytic phenotype of breast and ovarian cancer, and assess the possibility of exploiting several glycolytic targets therapeutically. Initially the growth dependency of breast and ovarian cancer cells on the availability of glucose was established. An array of 10 compounds reported to inhibit key enzymes of the glycolytic pathway were investigated and compared against an extended panel of breast and ovarian cancer cell line models. All inhibitors investigated, targeted against multiple points of the pathway, were shown to block the glycolytic pathway as demonstrated by glucose accumulation in the culture media combined with decreased lactate secretion, and attenuated breast and ovarian cancer cell proliferation in a concentration dependent manner. Furthermore their mechanism of action was investigated by flow cytometric analysis and their antiproliferative effect was associated with induction of apoptosis and G0/G1 cell cycle arrest. The glycolytic inhibitors were further assessed in combination strategies with established chemotherapeutic and targeted agents and several synergistic interactions, characterised by low combination index values, were revealed. Among them, 3PO (a novel PFKFB3 inhibitor) enhanced the effect of cisplatin in both platinum sensitive and platinum resistant ovarian cancer cells suggesting a strategy for treatment of platinum resistant disease. Furthermore robust synergy was identified between IOM-1190 (a novel GLUT1 inhibitor) and metformin, an antidiabetic inhibitor of oxidative phosphorylation, resulting in strong inhibition of breast cancer cell growth. This combination is proposed for the treatment of highly aggressive triple negative breast tumours. An additional objective of this research was to investigate the effect of the oxygen level on sensitivity to glycolysis inhibition. Breast cancer cells were found to be more sensitive to glycolysis inhibition in high oxygen conditions. This enhanced resistance at low oxygen levels was associated with upregulation of the targeted glycolytic enzymes as demonstrated at both the mRNA (by gene expression microarray profiling, Illumina BeadArrays) and protein level (by Western blotting). Manipulation of LDHA (Lactate Dehydrogenase A) by siRNA knockdown provided further evidence that downregulation of this target was sufficient to significantly suppress breast cancer cell proliferation. Finally, the expression of selected glycolytic targets was examined in a clinical tissue microarray set of a large cohort of ovarian tumours using quantitative immunofluorescence technology, AQUA. The role of the glycolytic phenotype in ovarian cancer was suggested and interesting associations between the glycolytic profile and clear cell and endometrioid ovarian cancers revealed. Increased PKM2 (Pyruvate kinase isozyme M2) and LDHA expression were demonstrated in clear cell tumours and also low expression of these enzymes was significantly correlated with improved survival of endometrioid ovarian cancer patients. Taken together the findings of this study support the glycolytic pathway as a legitimate target for further investigation in breast and ovarian cancer treatment.
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Lautru, Sylvie. "Purification and characterization of the glycolytic enzymes hexokinase and pyruvate kinase from Eurosta solidaginis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0035/MQ27053.pdf.
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Tunio, Sarfraz Ali. "Molecular and immunological characterization of glycolytic enzymes : FBA and GAPDH-1 of Neisseria meningitidis." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/13387/.
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There is growing evidence that several glycolytic enzymes, so-called housekeeping enzymes, including fructose bisphosphate aldolase (FBA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), despite being devoid of any apparent secretion signal, may be localised to the cell surface of several bacterial and fungal species, where they exhibit diverse non-glycolytic biological functions. However, the mechanism(s) of secretion of such signal-less proteins to the cell surface or to external environment is not well understood. Whilst their intracellular functions are well known, it is unclear whether they perform any additional functions, unconnected to their central role in glycolysis, on the bacterial surface. It is becoming apparent that such proteins may be immunogenic and they may be capable of eliciting protective immunity in animal models. As such, they represent potential vaccine candidates. In a search for novel surface exposed proteins as potential vaccine candidates against N. meningitides serogroup B, and in accordance with the fact that glycolytic enzymes are putative virulence factors in some bacterial species, it is hypothesised that meningococcal FBA and GAPDH-l, may be present on the cell surface and thus may contribute to the pathogenesis of disease. In N. meningitidis serogroup B, there is a single gene cbbA (NMB 1869) and two genes gapA-l and gapA-2, predicted to encode fructose bisphosphate aldolase and glyceradehyde 3-phosphate dehydrogenase (GAPDH) enzymes, respectively. Sequence analysis shows that FBA and GAPDH-l are highly conserved at the amino acid level. The amino acid sequences of FBA from N. meningitidis and those from Xanthobacter flavus and Synechocystis sp. displayed high identities (67 and 65%, respectively), which suggests that the meningococcal FBA (like those of X flavus and Synechocystis sp.) belongs to bacterial Class-II FBP aldolases. The cbbA and gapA-l genes were cloned and over-expressed in host E. coli. FBA was purified under non-denaturing and denaturing conditions, whilst GAPDH-l was purified under denaturing conditions. Recombinant native FBA was used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. The purified FBA and GAPDH-l proteins were then used to raise polyclonal monospecific rabbit antiserum (RaFBA and RaGAPDH-l) for subsequent characterisation of enzymes with the aim to determine their subcellular localization as well as potential roles in pathogenesis of meningococcal disease. RaFBA and RaGAPDH-l reacted with ca. 38-kDa and 37-kDa proteins, respectively, in immunoblot analysis against whole cell lysates from meningococcal strain MCS8 but not the cbbA and gapA-l isogenic mutants, respectively, confirming that cbbA and gapA-l are naturally-expressed proteins in N. meningitidis. Furthermore, expression of cbbA was detected in 26/26 and GAPDH-l in 17/17 diverse meningococcal strains. Cell fractionation experiments showed that meningococcal FBA and GAPDH-l are localized both to the cytoplasm and to the outer membrane. These results were validated by flow cytometry. The data demonstrated that outer membranelocalized FBA was surface-accessible to FBA-specific antibodies in encapsulated N. meningitidis, whereas flow cytometry analysis confirmed that GAPDH-I could be detected on the cell surface, but only in a siaD-deficient background, suggesting that GAPDH-I is inaccessible to antibody in encapsulated meningococci. Mutational analysis and functional complementation was used to identify additional functions of FBA and GAPDH-l. The cbbA and gapA-l knock-out mutant strains were unaffected in their ability to grow in vitro, but showed a significant reduction in adhesion to HBME and HEp-2 cells compared to their isogenic parent and complemented derivatives. In a transgenic mouse model, cbbA mutant strains were shown to be less able to establish bacteraemia compared to their wild-type parent strains. In summary, in this study, expression of FBA and GAPDH-l was shown to be well conserved across diverse isolates of Neisseria species. This study also demonstrates for the first time that meningococcal glycolytic enzymes, FBA and GAPDH-l. are surface localised proteins and required for optimal adhesion of meningococci to host cells. Taken together, these results suggest that FBA and GAPDH-l may be involved in the pathogenesis of meningococcal disease.
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Lautru, Sylvie (Sylvie Annie Jocelyne) Carleton University Dissertation Chemistry. "Purification and characterization of the glycolytic enzymes hexokinase and pyruvate kinase from Eurosta solidaginis." Ottawa, 1997.
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Zhong, Wenhe. "Biochemical and structural studies on trypanosomatid pyruvate kinases." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/7725.
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Glycolytic enzymes have been indicated as potential drug targets in trypanosomatid parasites such as Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania spp. Pyruvate kinase (PYK) catalyses the final reaction in the glycolytic pathway to produce ATP and pyruvate from ADP and phosphoenolpyruvate (PEP), and has been validated by RNAi experiments as a suitable drug target in T. brucei. This thesis describes biochemical and structural studies of PYKs from T. cruzi (TcPYK) and T. brucei (TbPYK), providing not only a foundation but also new clues for PYK-specific inhibitor screening and structure-based drug design. Soluble TcPYK and TbPYK (81% sequence identity) have been expressed and purified from E. coli, and their kinetics have been fully characterised. X-ray crystal structures of apoenzyme TcPYK (apo TcPYK), and of TbPYK in complex with fructose 2,6-bisphosphate (F26BP) (TbPYK/F26BP/Mg) have been determined, and each possesses a tetrameric architecture composed of four identical protein chains. Each chain contains four domains which are A-domain, B-domain, C-domain and N-terminal domain. The active site is located in the cleft between the A- and B-domains, while the F26BP-bound effector site is within the C-domain. The conformational transition between inactive T-state and active R-state for both enzymes requires a concerted 8o rigid-body rotation of each of the four AC-cores (Aand C-domains) in the tetramer. During the T- to R-state transition induced by F26BP binding, the side chain of Arg311 is re-orientated to stabilise the short Aα6′ helix at the active site, and the flexible loop at the effector site is stabilised by F26BP. In this active conformation additional salt bridges form across the C-C interface to lock the enzyme in a more stable R-state. TbPYK/F26BP/Mg is the first ‘effector only’ PYK structure and identifies a third Mg2+ binding site (Mg-3) which is distinct from the two canonical Mg2+ binding sites. The substrate PEP was soaked into crystals of TbPYK/F26BP/Mg resulting in an ‘in crystallo’ 23° B-domain rotation forming a partially closed active site. This is accompanied by active site side-chain reorientations, and the movement of Mg2+ from its ‘priming’ position Mg-3 to its canonical position Mg-1. It is plausible that Mg2+ is retained in its ‘priming’ position after product release to act as a co-activator with F26BP to maintain the enzyme in its R-state conformation, as long as F26BP is present. The inherent oxaloacetate decarboxylase activity of PYK was reported over 30 years ago and has been further characterised by 1H NMR studies in this thesis. In addition, a series of TbPYK structures in complex with product (pyruvate), with analogues of the decarboxylase substrate oxaloacetate (D-malate and α-ketoglutarate), or with the competitive inhibitor oxalate have been determined by crystal soaking, and indicate that both decarboxylase activity and kinase activity share a common active site. A proposed mechanism explains the conserved decarboxylase activity of PYK where the active-site Mg2+ and Lys239 in TbPYK (which is conserved between species) play essential roles in the decarboxylation reaction. Three strategies for designing novel inhibitors against trypanosomatid PYKs have been proposed in this thesis. (1) Develop selective modulators to increase the binding affinity of inhibitors. As an example, F16BP has been shown to regulate the inhibitory effect of PEP analogues (oxalate, D-malate, α-ketoglutarate, malonate and L-tartrate) on TbPYK activity. (2) Develop allosteric inhibitors in order to lock trypanosomatid PYKs in an inactive state where the enzyme has low affinity for substrate binding. (3) A third strategy is to combine multiple modulators and inhibitors to increase the inhibition efficiency and selectivity.
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Gamieldien, Kareemah. "The influence of maternal nicotine exposure on selected glycolytic and cytochrome P450 enzymes in developing neonatal rat lung." Thesis, University of the Western Cape, 2005. http://etd.uwc.ac.za/index.php?module=etd&.
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The structural and functional integrity of a developing and maturing fetal and neonatal lung is critically dependent on carbohydrate metabolism. The energy derived from carbohydrate metabolism is utilized during the processed of cell growth and development. It is reported that maternal nicotine exposure during pregnancy and lactation results in the irreversible inhibition of glycolysis, for which no mechanism is currently proposed and a significant increase in glucose turnover. The principal objective of this thesis was to determine the influence of maternal nicotine exposure during gestation and lactation on the isoenzyme patterns and transcript levels of the selected enzymes in developing neonatal rat lung, in an attempt to elucidate the mechanism of inhibition of glycolysis observed.
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Veivers, Pamela Christine. "Biochemical aspects of symbiosis in carbon and nitrogen metabolism in higher termites." Thesis, The University of Sydney, 1995. https://hdl.handle.net/2123/26814.
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A comprehensive study of carbon metabolism has been carried out on representatives of two genera of higher termites, Nasutitermes walkeri and the fungus gardeners, Macrotermes spp. Substantial activity of cellulase and its components endo-B-l,4—glucanase and B—glucosidase, was found in workers of N. walkeri, Macrotermes michaelseni M. subhyalinus, M. bellicosus and M. mulleri. Less activity was found in the soldiers and none in the larvae of the Macrotermes spp. In fungal material associated with Macrotermes spp. only the fungal nodules had significant enzyme activity. In N. walkeri and M. michaelseni at least 90% of all enzyme activities was found in the midgut, with the exception of endo-B-l,4-xy1anase activity which was not measured in M. michaelseni. Low levels of endo-fi-l,4—xylanase activity were evenly distributed throughout the gut of N. walkeri. Midgut enzyme activity was restricted to the anterior region. The salivary glands contained variable amounts of enzyme activities. Less than 5% of the endo-B-l,4— glucanase activity was located in the hindgut. The endogenous cellulase from N. walkeri consisted of multiple B-glucosidase and endo-B-1,4- glucanase components. Elution profiles of fungal endo-B—l,4—glucanase activities on Bio-Gel® P-150 indicate that each Macrotermes spp. has a different Termitomyces sp. associated with it, consisting of one to three endo-B-l,4-glucanase activities. Endo-B-1,4-glucanase components from termite workers also consisted of one to three enzymes. A comparison of the elution profiles on Bio-Gel® P-60 of endo-B-lA—glucanase and Bglucosidase activities from fungal and termite material was used to show the absence of fungal enzymes in the termite gut. Similar results were found with fungal and termite material from M. bellicosus and M. mulleri using Bio-Gel® P—150 chromatography.
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Zondi, Nondumiso Busisiwe Ntombikhona. "Effect of heat stress of the cell wall of the yeast, Saccharomyces cerevisiae : phosphorylation of ribosomal protein S10-B brought about by enzymes of the glycolytic pathways." Master's thesis, University of Cape Town, 2005. http://hdl.handle.net/11427/4354.
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Includes bibliographical references.The cell wall is a dynamic and elastic structure that provides osmotic and physical protection to the yeast cell (l). As such, it forms the immediate site of contact between the yeast cell and its environment (2), and is essential for maintaining cell integrity and shape. Cell growth and development demand that the cell wall is not rigid and unchangeable as the cell needs to adjust the wall composition and structure during growth and development stages such as morphogenesis, flocculation, cell-cell recognition and pathogenicity, and in response to changes in environmental conditions (l, 2, 3, 4).
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Daryaei, Hossein, and s3088498@student rmit edu au. "Application of high pressure processing for extending the shelf-life of fresh lactic curd cheese." RMIT University. Applied Science, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080821.155923.
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Outgrowth of spoilage yeasts and moulds and post-processing acidification can limit the shelf-life of some fermented dairy products including fresh lactic curd cheeses. The possibility of using high pressure processing (HPP) for controlling these problems was investigated in a commercially manufactured fresh lactic curd cheese (pH 4.3-4.4) and fermented milk models (pH 4.3-6.5). The effects of HPP at 300 and 600 MPa on inactivation of glycolytic enzymes of lactic acid bacteria were also evaluated. Fresh cheeses made from pasteurised bovine milk using a commercial Lactococcus starter preparation were treated with high pressures ranging from 200 to 600 MPa (less than or equal to 22°C, 5 min) under vacuum packaging conditions and subsequently stored at 4°C for 8 weeks. Treatment at greater than or equal to 300 MPa substantially reduced the viable count of Lactococcus and effectively prevented the outgrowth of yeasts and moulds for 6 to 8 weeks without adversely affecting the sensory and textural attributes of the product. However, it had no significant effects (p less than 0.01) on variation of titratable acidity during storage. Fermented milk models were prepared by individually growing Lactococcus lactis subsp. lactis C10, Lactococcus lactis subsp. cremoris BK5, Streptococcus thermophilus TS1, Lactobacillus acidophilus 2400 and Lactobacillus delbrueckii subsp. bulgaricus 2517 in UHT skim milk and diluting the resulting fermented milk with UHT skim milk up to pH 6.5. Pressure treatment of the milk models at pH 5.2 resulted in substantial inhibition of post-processing acidification during storage and markedly reduced the viable count of Lactococcus at both 300 and 600 MPa and other bacteria only at 600 MPa. Treatment of the milk model at 600 MPa decreased the viable counts of Candida zeylanoides and Candida lipolytica (wildtype spoilage yeasts of lactic curd cheese, added as challenge cultures) from 105 CFU mL-1 to below the detection limit (log 0 CFU mL-1) at all pH levels tested (pH 4.3-6.5) and effectively controlled their outgrowth for 8 weeks. Treatment of milk model at 300 MPa had a similar effect only on C. zeylanoides. The viable count of C. lipolytica was reduced by 2.6, 2.4 and 2.3 logs by treatment at 300 MPa at pH levels of 4.3, 5.2 and 6.5, respectively, which subsequently recovered by 2.9, 2.8 and 3.2 logs within 3 weeks. Glycolytic enzymes of various starter bacteria showed different responses to pressure treatment. The lactate dehydrogenase in L. lactis subsp. lactis and Lb. acidophilus was quite resistant to pressures up to 600 MPa, but it was almost completely inactivated in S. thermophilus at pressure levels as low as 300 MPa. The â-galactosidase in Lb. acidophilus was more pressure stable than â-galactosidase in S. thermophilus and Phospho-â-galactosidase in L. lactis subsp. lactis. The findings of this study suggests HPP at 300-600 MPa as an effective method for controlling the outgrowth of some spoilage yeasts and moulds in fresh lactic curd cheeses. The results obtained with selected lactic acid bacteria in fermented milk models can be used to assist in establishing HPP operating parameters for development of new generation cultured dairy products, of reduced acidity and extended shelf-life.
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McHarg, Jane. "Investigations into the glycolytic enzyme phosphoglycerate kinase." Thesis, University of Exeter, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390169.
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Tomotani, Ester Junko. "Bioconversão de sacarose em ácido glicônico e frutose usando reator com membrana." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/9/9134/tde-16082017-123204/.
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A conversão enzimática da sacarose pela ação sucessiva da invertase e da glicose oxidase (GOD), permite obter produtos de maior valor agregado, a saber, frutose e o ácido glicônico, dois produtos de amplo uso na indústria farmacêutica, alimentícia e química. Foi estudada a aplicação da invertase imobilizada em resinas aniônicas do tipo Dowex® (um copolímero de poliestireno-divinilbenzeno) sobre a hidrólise da sacarose bem como a oxidação da glicose pela glicose oxidase solúvel ou imobilizada no mesmo suporte em separado (sistema bifásico), utilizando-se um reator de membrana acoplado à membrana de ultrafiltração (100kDa) ou de microfiltração (5µm). Posteriormente, avaliou-se o desempenho de ambas as formas de enzimas, solúveis ou imobilizadas num sistema monofásico empregando o mesmo reator. A bioconversão executada em sistema bifásico permitiu a obtenção de xarope de frutose da ordem de 70% através da separação de glicose e frutose utilizando-se a resina catiônica 50W:8-100. O rendimento de 96,6% e 67,4% para as formas solúveis e imobilizadas respectivamente foram obtidas em sistema monofásico. O não desprendimento das enzimas dos suportes viabilizou o uso da membrana de microfiltração, trazendo vantagens à operação de biorreator com membrana.The enzymatic conversion of sucrose through a successive action of invertase and glucose oxidase (GOO) allows the obtainment of products with higher commercial value, fructose and gluconic acid, which are widely used in pharmaceutical, food and chemical industries. Invertase and GOO immobilized on Dowex® anionic resin (a polystyrene divinylbenzene copolymer) as well as soluble GOD were used in a membrane bioreactor (MS) for sucrose hydrolysis and glucose oxidation. The MB was coupled with a UF-membrane (100kDa) or a MF-membrane (5µm). The bioconversion was conducted in two steps (biphasic system) as well as in one step (monophasic system). The bioconversion operated in a biphasic system permitted obtaining a fructose syrup with a concentration of about 70% through a separation of glucose and fructose using a cationic resin, 50W:8-100. As for the monophasic system, the yield of 96.6% and 67.4% for soluble and immobilized forms were attained respectively. No leakage of the enzymes from the support allowed the use of a microfiltration membrane, adding advantages to the membrane bioreactor operation.
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Frodsham, G. "A study of glycolytic enzyme distribution and transient times in heart." Thesis, University of Liverpool, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370845.
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Yang, Min. "Synthesis of 5 thioglucose derivatives in the carbohydrate metabolic pathways in lactic acid bacteria." Thesis, University of Huddersfield, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274227.
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Blount, Kathryn. "Cancer systems biology : is the devil in the glycolytic detail?" Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/cancer-systems-biology-is-the-devil-in-the-glycolytic-detail(e0ad0c6b-76ec-4bba-8dd3-b583910f46f4).html.
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An approach to investigating cancer that has recently seen resurgence of interest is the “Warburg effect”. Otto Warburg originally described the altered metabolism of cancer cells and identified that they exhibit an increase in glucose uptake and lactate production. This up-regulation of glycolytic flux and glucose transport is now associated with 90% of cancers. In order to improve the overall understanding of the “Warburg effect” two forms of systems biology have been implemented - comparative in vitro analysis of kinetic activities and dynamic modelling. In this analysis, human breast cancer cell lines MCF-7, MDA-MB-231 and T47D and a non transformed breast cell line MCF-10A were used to identify key similarities and differences in kinetic activities across the glycolytic pathway. Additionally, activities of key glycolytic enzymes hexokinase, pyruvate kinase and lactate dehydrogenase were compared under hypoxic conditions to further understand regulation of cancer cells. The most prominent feature that arose from comparing the kinetic activities of the three malignant and one non-malignant cell line is that each cell line has its own specific set of activities for glycolysis. This indicates that there are differences in regulation across the glycolytic pathway for each of these cell lines. This is of specific interest in the search for therapeutic targets. Further, we determined that despite the prominence of oncogenic HIF signalling activities of hexokinase, pyruvate kinase and lactate dehydrogenase were further modulated by growth under hypoxic conditions. Despite the lack of obvious distinct kinetic differences between the non-cancerous and cancerous cells lines some discernible differences are apparent when modelled in silico.
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Stevenson, Jack Alan. "Copper binding of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase in Staphylococcus aureus." Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3505.
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Staphylococcus aureus is a Gram positive bacterium which is predominantly commensal, but which can also be a human and animal pathogen, able to cause serious infections. It is becoming an increasing problem due to it becoming resistant to modern antibiotics. In common with most bacteria, S. aureus requires small quantities of the essential metal copper, but they also experience toxicity when exposed to high concentrations of copper, a metal that has been used for its antimicrobial properties for centuries. However, the mechanism of such toxicity remains elusive. Here, the effect of copper toxicity on S. aureus has been investigated in order to understand how excess copper ions affect its physiology. The growth of S. aureus was found to be inhibited in media containing elevated copper, and the extent of this inhibition was shown to be dependent on the type of growth medium used. Analysis of soluble extracts from S. aureus cells exposed to elevated copper led to the identification of a cytoplasmic enzyme, GapA, which binds copper. GapA is a member of the wellcharacterised family of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes, and is not a metalloprotein. Copper inhibition of the activity of the S. aureus GapA enzyme was demonstrated, both in S. aureus cell extracts and with recombinant enzyme in vitro, using a specific enzyme activity assay. Analysis of the purified recombinant GapA enzyme in vitro demonstrated a copper binding stoichiometry of one Cu(I) ion to each GapA monomer using analytical sizeexclusion chromatography and spectrophotometry, and evidence is presented that suggests high affinity Cu(I) binding of biochemical relevance. Some preliminary evidence is provided that the Cu(I) binding site on the GapA protein includes the thiol group of cysteine 151 located within the active site, consistent with predictions based on published crystal structures, and explaining the observed copper-dependent inhibition of enzyme activity. This is the first evidence of copper binding to a non-metalloprotein within the cytoplasm of S. aureus and adds to mounting evidence that aberrant binding of copper to cytosolic proteins contributes to bacterial copper toxicity.
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Rypniewski, W. R. "The structure of unliganded E. coli phosphofructokinase." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233316.
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The allosteric phosphofructokinase from E. coli crystallized in the absence of ligands. X-ray diffraction data were collected to 2.4 aa, AA resolution and the structure was solved by the method of molecular replacement, using the model of the liganded R-state of the enzyme. The atomic model was refined by rigid body refinement and by the least-squares method of Hendrickson and Konnert. The final structure is compared to the high resolution model of the liganded, active form of the enzyme and to the low resolution structure of the inhibited phosphofructokinase from B. stearothermophilus. It is found that the quaternary structure of the unliganded model is more similar to the liganded, active form than to the inhibited T-state of B. stearothermophilus pfk. There are however considerable differences in the tertiary and quaternary structures, apparently resulting from ligand binding. These changes are not localised to the binding sites. The overall change, though to result mainly from the binding of the activators, is consistent with the closing of the active site observed in the structure of the active state. The ways in which the ligand binding could bring about these changes are considered. The possible effect of the changes on the enzyme activity are discussed. It is possible that the structure represents an inactive T-state, different from that in the presence of inhibitors. Whatever the exact interpretation it is clear that the structure shows considerable flexibility suggesting that the two-state allosteric model is an oversimplification.
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Arya, Roopen. "Molecular basis of tiosephosphate isomerase deficiency." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321940.
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Hartman, Angela Danielle. "Effect of Metabolic Enzymes on Amylopectin Content and Infectivity of Cryptosporidium parvum." Diss., Virginia Tech, 2006. http://hdl.handle.net/10919/29862.
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Amylopectin granules in Apicomplexan protozoa are hypothesized to be used as an energy source to aid the parasites in surviving in the environment allow latent stages to excyst and release infective stages, allow them to be mobile, invade host cells, and to continue their life cycle. The objective of this project was to determine if parasite glycolytic enzymes: alpha-amylase, amyloglucosidase, enolase, lactate dehydrogenase, and phosphorylase could be used to decrease amylopectin stores and subsequently infectivity of Cryptosporidium parvum oocysts/sporozoites in both fresh oocysts and stored oocysts. In addition, glycolytic enzymes and substrates: glucose, glucose-1-phosphate, and glycogen synthase were investigated to determine if they can be used to increase amylopectin stores and thus increase infectivity to aid in detection/storage of oocysts. Oocysts of Cryptosporidium parvum were suspended in 1mg/ml glycolytic enzymes or substrates (except glucose - 0.05M and glycogen synthase - 1U/ml) and electroporated. Oocysts were incubated at 37°C for one hour to allow treatments to react with amylopectin followed by incubation on HCT-8 cells for 24 hours for infection. Real-time PCR and immunohistochemistry were performed to determine the effect of the enzymes on infectivity. An amylopectin assay and excystation assay was performed to determine if the enzymes degraded amylopectin and if decreased amylopectin reduced excystation. Alpha amylase and amyloglucosidase had the greatest impact on reducing both amylopectin and infectivity of fresh oocysts with reductions of 99.5% and 99.1% in infective oocysts, respectively (P<0.05). These results suggest that amylopectin may be an important factor in infection, although further research is needed. In stored oocysts, enzymes significantly reduced amylopectin content but not infectivity. In fresh oocysts, amylopectin content was correlated to excystation and infectivity with a decrease in amylopectin correlating to decreased excystation and infectivity. In contrast, there was no direct correlation for stored oocysts. When glucose, glucose-1-phosphate, or glycogen synthase was used to increase infectivity, results show that glycogen synthase had little effect, but glucose and glucose-1-phosphate significantly increased amylopectin content, excystation, and infectivity. In conclusion, amylopectin may be an important polysaccharide store of Cryptosporidium parasites to cause infection by allowing excystation of the oocysts to release infective sporozoites.Ph. D.
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Horemans, Steff. "Discovery of a new type of regulator of DNA replication : the glycolytic enzyme pyruvate kinase in Bacillus subtilis." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLE040.
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On sait depuis longtemps que le fenêtrage de la réplication de l'ADN dans le cycle cellulaire chez les bactéries varie avec la richesse en nutriments. Cependant, le mécanisme sous-jacent reste pour l’instant inconnu. Le but de ce travail est de déterminer si PykA, une enzyme du métabolisme central carboné (MCC), est impliquée dans ce contrôle temporel de la réplication chez la bactérie Bacillus subtilis et si, pour assurer cette fonction, PykA utilise une activité non-métabolique cryptique qui lui permet d’agir en tant qu'effecteur temporel de la réplication en transmettant des informations sur l'état métabolique de la cellule à l'appareil de réplication.Pour tester ces hypothèses, l’effet de mutations dans le gène pykA sur la réplication a été évalué dans un milieu de culture où son activité métabolique n’est pas nécessaire. Les paramètres de réplication dans ces mutants ont alors été mesurés par des approches de cytométrie en flux et de PCR quantitative.Nos résultats ont confirmé que la délétion de pykA n’a pas d’effet sur la croissance des cellules dans le milieu utilisé mais que cette mutation affecte le contrôle temporel de la réplication en modifiant l’âge d’initiation de la réplication et la vitesse de réplication. La délétion du domaine catalytique ou du domaine « PEP utilizer » de PykA a montré que chacun de ces domaines joue un rôle dans le contrôle temporel de la réplication, assignant pour la première fois une fonction de réplication à ces domaines. Une mutagenèse ciblée du domaine catalytique a montré que seulement certains acides aminés impliqués dans l’activité métabolique de PykA sont importants pour la réplication. La mutagenèse des acides aminés conservés dans le motif Thr-Ser-His (TSH) du PEP utilizer suggère un rôle important de la thréonine dans le contrôle temporel de la réplication. L'expression du PEP utilizer seul (non fusionné au domaine catalytique), a permis de découvrir plusieurs principes de réciprocité entre les deux domains. Enfin, des tests in vitro ont démontré que PykA peut directement stimuler l'activité de la polymérase DnaE et inhiber l'activité de l’hélicase DnaC. Ensemble, ces résultats indiquent que PykA a une activité cryptique de réplication.Pour démontrer que PykA transmet l'état métabolique de la cellule à la machinerie de réplication, l'effet de mutations dans pykA sur les paramètres de réplication de cellules subissant un changement métabolique a été évalué à l'aide des techniques décrites ci-dessus.Nous avons observé que l’adaptation des paramètres de réplication au cours du changement métabolique dans les cellules sauvages se déroule en trois phases: (i) une diminution progressive de la vitesse de la fourche de réplication de 15 à 45 minutes après le changement métabolique, (ii) une stimulation de l’initiation à partir du point 30 minutes et (iii) finalement une adaptation du taux de croissance après 45 minutes. Les mutations dans le domaine catalytique ne semblent pas avoir d’effet sur l’évolution de ces paramètres. En revanche, le PEP utilizer, fusionné ou non au domaine catalytique, affecte l’adaptation des paramètres de réplication par un processus nouveau dépendant de l'acide aminé His et non Thr du motif TSH. Ainsi donc, le PEP utilizer et plus généralement PykA, agit sur la réplication selon des voies particulières en fonction du métabolisme.Nous concluons que PykA est un nouveau type de régulateur de la réplication de l’ADN qui facilite le positionnement temporel de la réplication par le biais de processus variant avec la richesse des sources de carbone fournies dans l’environnement. Ces travaux, combinés à des travaux antérieurs dans ce domaine, suggèrent que le contrôle temporel de la réplication dans un large éventail de conditions de croissance métabolique est réalisé au moins en partie via un réseau d'enzymes MCC ayant des activités cryptiques. Cette conclusion a des implications importantes dans notre compréhension de la biologie cellulaireIt has been known for decades that the timing of DNA replication within the cell cycle in bacteria is coupled to nutrient richness. However, the mechanisms that enable this temporal gating have remained elusive. The goal of this work was to test whether the CCM enzyme PykA in the bacterium B. subtilis helps achieve this gating by moonlighting as a temporal effector of replication that communicates information about the metabolic state of the cell to the replication machinery.To demonstrate that PykA moonlights as a replication protein, the effect of pykA mutations on DNA replication parameters was assessed using run-out flow cytometry and marker frequency analysis by qPCR in growth conditions where its metabolic activity is dispensable.Deletion of pykA did not affect the growth rate during these conditions, but it affected the replication speed and timing of initiation, altering therefore the temporal control of replication. The deletion of either the catalytic domain or the PEP utilizer domain of PykA showed that both domains play a role in the temporal control of replication, identifying for the first time a replication function to these domains. Mutagenesis of specific amino acids inside the catalytic domain showed that some, but not all key catalytic amino acids are involved in replication. Mutagenesis of conserved amino acids in the TSH motif of the PEP utilizer on the other hand suggests an important role for threonine phosphorylation. Expression of the PEP utilizer detached from the catalytic domain in trans unexpectedly affected replication control at medium copy numbers. This phenotype depended strictly on H of the TSH motif, establishing a role for H in replication. Furthermore, the effect of expression of free PEP utilizer may depend of an unidentified inhibitor effector and is fully suppressed by a mutation in the catalytic domain. Moreover, expression of the catalytic domain in cis and the PEP utilizer in trans did not restore proper replication control. Finally, in vitro assays demonstrated that PykA can directly stimulate DnaE polymerase activity and inhibit DnaC helicase activity. Together, these results imply that PykA moonlights in replication.To demonstrate that PykA communicates information about the metabolic state of the cell to the replication machinery, the effect of pykA mutations on DNA replication parameters during a metabolic shift was assessed using the techniques described above.The adaptation of replication parameters during the metabolic shift in wild type cells unexpectedly consisted of three phases: (i) a decrease of replication fork speed between 15 and 45 min after the shift, (ii) an advancement of initiation timing and increase in initiation frequency from 30 min onwards and (iii) an adaptation of the growth rate sometime after 45 min after the shift. Mutations in the catalytic domain did not clearly demonstrate a change in the replication response to the shift. By contrast, the effect of the PEP utilizer domain (both in cis and in trans) on replication parameters did change during the shift. Moreover, the H amino acid, not T, was important for replication control during later time points of the shift. This suggests that the regulatory behaviour of the PEP utilizer and more generally PykA changes in response to metabolism.Overall, we conclude that PykA is a new type of regulator of DNA replication that helps the temporal positioning of replication in the cell cycle through processes that varies with the richness of the carbon sources provided in the environment. This work, combined with previous work in this field suggest that the temporal control of replication in a large range of metabolic growth conditions is achieved at least in part via a network of moonlighting CCM enzymes with important implications for our basic understanding of cell biology and human health
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Quinn, Gregory Bernard. "The recombinant expression and characterization of human neuron specific enolase." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241344.
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Okuda, Junji. "Persistent Overexpression of Phosphoglycerate Mutase, a Glycolytic Enzyme, Modifies Energy Metabolism and Reduces Stress Resistance of Heart in Mice." Kyoto University, 2014. http://hdl.handle.net/2433/185197.
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Eicher, Johann Josef. "Understanding glycolysis in Escherichia coli : a systems approach using nuclear magnetic resonance spectroscopy." Thesis, Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/85730.
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Thesis (PhD)--Stellenbosch University, 2013.ENGLISH ABSTRACT: This dissertation explores the behaviour and regulation of central carbon metabolism in Escherichia coli K12 W3110 under fermentative microaerobic conditions. To achieve this, an integrative systems modelling approach was adopted, which is introduced in Chapter 1 along with a review of metabolism in E. coli. An open-source software suite NMRPy, developed using the Python programming language, is presented in Chapter 2. NMRPy provides a host functions for basic processing, analysis and visualisation of Nuclear Magnetic Resonance (NMR) spectroscopy data. In addition to this, NMRPy offers specialised functions for the deconvolution of arrayed reaction time series, which proved indispensable to the research presented in this dissertation. NMRPy presents an easy to use, extensible tool for both routine and advanced use. In Chapter 3, a novel methodology is presented which was developed for the effective and comprehensive determination of enzyme kinetic parameters for systems biology using NMR. In contrast to traditional enzyme kinetic assay methods, this new methodology is less labour-intensive and yields significantly more information per experiment. By fitting kinetic equations to real time NMR data, dynamic changes in substrates, products and allosteric modifiers are quantified and allowed to inform the parameter fitting procedure. These data contain information on cooperative substrate binding, reversibility, product inhibition and allosteric effects. The proposed methodology is applied to the study of the first two enzymes of the glycolytic pathway. In Chapter 4, the construction, parameterisation and validation of a number of kinetic models of glycolysis in E. coli under microaerobic conditions is detailed. To model the lower half of glycolysis, a similar technique was adopted as in Chapter 3, in which models representing the reactions from triosephosphate isomerase to pyruvate kinase were parameterised by fitting them to a collection of 31P NMR reaction time series. This approach extends the methodology to enzyme sub-networks, yielding data that encompass the full complexity of the network regulatory interactions. The verified kinetic models were subjected to scrutiny, the results of which are presented in Chapter 5. The value of the modelling approach is demonstrated by the ease with which cumbersome in vivo experiments can be performed in silico. A structural analysis of the model topology was conducted, elucidating the elementary flux modes of fermentative glycolysis in E. coli, and identifying a futile cycle around PEP carboxylase and PEP carboxykinase. Model steady-state behaviour and control properties were explored in silico under various degrees of ATP demand and oxygen availability and a number of hypotheses are presented, explaining the regulation of free energy in E. coli, and the metabolic responses of E. coli to changing redox demands. Amongst other things, the results demonstrated that the glucose importing phosphoenolpyruvate: phosphotransferase pathway controlled glycolytic flux, and that under microaerobic conditions E. coli is able to regulate redox balance not only by balancing flux between acetate and ethanol, but also by altering the balance of flux between acetate and lactate at the pyruvate formate lyase/lactate dehydrogenase branch point. This study demonstrates the value of an integrated computational and experimental systems approach to exploring biological phenomena.
AFRIKAANSE OPSOMMING: In hierdie proefskrif word die gedrag en regulering van die sentrale koolstofmetabolisme in Escherichia coli K12 W3110 onder fermenterende mikro-a¨erobiese toestande ondersoek. Dit is moontlik gemaak deur ’n ge¨ıntegreerde stelsel-modelleringsbenadering, wat in Hoofstuk 1 bekendgestel word. D´ıe hoofstuk verskaf ook ’n oorsig van die metabolisme in E. coli. ’n Oopbron-kodepakket NMRPy, wat in die programmeringstaal Python ontwikkel is, word in Hoofstuk 2 beskryf. NMRPy verskaf ’n aantal funksies vir die basiese verwerking, analise en visualisering van Kern-Magnetiese Resonansie (KMR) spektroskopiese data, sowel as gespesialiseerde funksies vir die dekonvolusie van opeenvolgende reaksie-tydreekse. Hierdie funksionaliteit was onontbeerlik vir die verdere navorsing in hierdie proefskrif. Hoofstuk 3 beskryf die ontwikkeling van ’n nuwe metodiek vir die omvangryke bepaling van ensiem-kinetiese parameters vir sisteembiologie, deur van KMR gebruik te maak. In teenstelling tot tradisionele ensiem-kinetiese essai-metodes, is hierdie nuwe metodologie minder arbeidsintensief en lewer dit beduidend meer inligting per eksperiment. Deur die kinetiese vergelykings op tydsafhanklike KMR data te pas, word dinamiese veranderinge in substrate, produkte en allosteriese effektors gekwantifiseer en hierdie inligting gebruik in die passingsprosedure. Die data bevat inligting oor ko¨operatiewe substraatbinding, omkeerbaarheid, produkinhibisie en allosteriese effekte. Die voorgestelde metodologie word toegepas op die karakterisering van die eerste twee glikolitiese ensieme. In Hoofstuk 4 word die konstruksie, parameterisering en validering van ’n aantal kinetiese modelle van glikolise in E. coli onder mikro-a¨erobiese toestande uiteengesit. Die waarde van die modelleringsbenadering lˆe in die gemak waarmee omslagtige in vivo eksperimente in silico uitgevoer kan word. Om die onderste helfte van die glikolitiese pad te modelleer word ’n soortgelyke tegniek as in Hoofstuk 3 gebruik. Modelle van die reaksies vanaf triosefosfaat-isomerase tot by pirovaat-kinase is geparameteriseer deur dit op ’n versameling 31P KMR-tydreekse te pas. Hierdie benadering brei bostaande metodologie uit tot ensiem-subnetwerke en genereer data wat die volle kompleksiteit van regulerende interaksies in die netwerk insluit. Die geverifieerde modelle word in Hoofstuk 5 noukeurig ondersoek. ’n Strukturele analise van die modeltopologie word onderneem om die elementˆere fluksie-modes van fermentatiewe glikolise in E. coli te verklaar, sowel as om ’n futiele siklus rondom fosfo¨enolpirovaat karboksilase en fosfo¨enolpirovaat karboksikinase te identifiseer. Die bestendige-toestandsgedrag en kontrole-eienskappe word in silico ondersoek onder toestande van verskeie ATP beladings en suurstofbeskikbaarheid. ’n Aantal hipoteses word voorgelˆe, wat die regulering van vry energie in E. coli, sowel as die metaboliese reaksies van E. coli onder veranderende redoks-vereistes kan verklaar. Onder andere dui die resultate daarop dat die fosfo¨enolpirovaat:fosfotransferase sisteem (wat verantwoordelik is vir glukose-opname in die sel) die glikolitiese fluksie beheer en dat E. coli onder mikro-a¨erobiese toestande die redoksbalans nie net tussen asetaat en etanol kan reguleer nie, maar ook die deur wysiging van die fluksie-balans tussen asetaat en laktaat rondom die pirovaat-formiaat-liase/laktaatdehidrogenase vertakkingspunt. Hierdie studie toon die waarde van ’n ge¨ıntegreerde rekenaarmatige en eksperimentele sisteembenadering om biologiese verskynsels te ondersoek.
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Hole, Rebecca. "Mammalian ADP-dependent glucokinase : a thesis presented in partial fulfilment of the requirement for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand." Massey University, 2009. http://hdl.handle.net/10179/1154.
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The mammalian ADP-dependent glucokinase is the most recent mammalian glucokinase to have been discovered, and is unique in its ability to catalyse the phosphorylation of glucose to glucose-6-phosphate using ADP as the phosphoryl donor. Up until the discovery of this enzyme, the traditional biochemical view was that the first step of glycolysis was solely catalysed by ATP-dependent hexokinases, types I-IV. The particular role played by ADP-GK in the mammalian cell and the significance of this role has not yet been determined, although it is hypothesised that the ADP-dependent glucokinase could be potentially significant in contributing to the survival of cells under low energy and hypoxic or ischemic conditions. By using ADP as the energy investment in phase one of the glycolytic cycle instead of ATP, it is predicted that glycolysis could be sustained for longer during lower energy conditions (conditions of high ADP:ATP ratios). Since the phosphorylation of glucose by ADP-GK results in the production of AMP, it may also be possible that this has a direct effect on the energy charge of the cell. The AMP produced could lead to the regulation of cellular metabolism during hypoxia and/or ischemia via the activation of the cell-energy regulator AMPK. The study of mammalian ADP-dependent glucokinase is a very new area, and prior to this no investigation of the human ADP-GK enzyme had been undertaken. The main objective of this project was to clone, express and purify the recombinant ADP-GK so it could be kinetically characterised and directly compared with the recombinant mouse kinetic characteristics, the only other mammalian ADP-GK to have been studied. Unfortunately, due to complications in the expression and purification of soluble recombinant human ADP-GK, the project did not incorporate the kinetic characterisation of the enzyme. Acquiring data on the kinetic characteristics of the human ADP-GK will, in the long term, assist in the elucidation of the metabolic role of this enzyme, so the continuation of this project would be worthwhile.
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Haferkamp, Patrick [Verfasser], Bettina [Akademischer Betreuer] Siebers, and Daniel [Akademischer Betreuer] Hoffmann. "Biochemical studies of enzymes involved in glycolysis of the thermoacidophilic crenarchaeon Sulfolobus solfataricus / Patrick Haferkamp. Gutachter: Daniel Hoffmann. Betreuer: Bettina Siebers." Duisburg, 2011. http://d-nb.info/1017931879/34.
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Eldib, Abdallah. "Proteomic and molecular studies on the human follice and follicular fluid : phenotypic differences between human granulosa cell subtypes with special reference to glycolytic enzyme expression." Thesis, University of Leeds, 2009. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509826.
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Acar, Seyda. "Biochemical And Genetic Studies On The Pyruvate Branch Point Enzymes Of Rhizopus Oryzae." Phd thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/3/12604762/index.pdf.
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Rhizopus oryzae is a filamentous fungi which produces lactic acid and ethanol in fermentations. R. oryzae has numerous advantages for use industrial production of L-(+)-lactic acid but the yield of lactic acid produced on the basis of carbon consumed is low.
Metabolic flux analysis of R. oryzae has shown that most of the pyruvate produced at the end of the glycolysis is channelled to ethanol, acetyl-CoA and oxaloacetate production. This study aimed to answer some questions addressed on the regulation of pyruvate branch point in R. oryzae and for this purpose biochemical characterisation of the enzymes acting at this branch point and cloning the genes coding for these enzymes have been done.
Pyruvate decarboxylase was purified and characterised for the first time from R. oryzae. The purified enzyme has a Hill coefficient of 1.84 and the Km of the
enzyme is 8.6 mM for pyruvate at pH 6.5. The enzyme is inhibited at pyruvate
concentrations higher than 30 mM. The optimum pH for enzyme activity shows a broad range from 5.7 and 7.2. The monomer molecular weight was estimated as 59±2 kDa by SDS-PAGE analysis. Pyruvate decarboxylase (pdcA and pdcB) and lactate dehydrogenase (ldhA and ldhB) genes of R. oryzae have been cloned by PCR-cloning approach and the filamentous fungi Aspergillus niger was transformed with these genes. The A. niger transformed with either of the ldh genes of R. oryzae showed enhanced production of lactic acid compared to wild type. Citric acid production was also increased in these transformants while no gluconate production was observed Cloning of hexokinase gene from R. oryzae using degenerate primers was studied by the use of GenomeWalker kit (Clontech). The results of this study were evaluated by using some bioinformatics tools depending on the unassembled clone sequences of R. oryzae genome.
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Duminil, Pauline. "Characterization of two primary metabolism enzymes in Arabidopsis thaliana : phosphoglycerate mutase and phosphoglycolate phosphatase." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS591.
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Les plantes sont des organismes sessiles. Elles doivent réagir rapidement et efficacement aux stress biotiques et abiotiques qu’elles subissent. Pour cela, elles utilisent plusieurs niveaux de régulation. L’un d’eux, rapide et réversible, consiste a effectuer des modifications post-traductionnelles (PTMs) sur ses enzymes. La PTM la plus répandue est la phosphorylation protéique, qui intervient dans diverses voies du métabolisme primaire. La glycolyse permet la production d’énergie (ATP) et de pouvoir réducteur à partir de glucose. La régulation de la phosphoglycérate mutase d’Arabidopsis thaliana (AtiPGAM) a été étudiée grâce à une analyse d’un site de phosphorylation dans le but d’élucider le mécanisme réactionnel. La photorespiration est un processus essentiel pour les organismes photosynthétiques. Ce cycle, initié par l’activité oxygénase de la ribulose-1,5-biphosphate carboxylase / oxygénase (RuBisCO), produit notamment une molécule de 2-phosphoglycolate (2-PG), toxique pour la plante. Le recyclage, couteux, du 2-PG par le cycle photorespiratoire se déroule dans quatre compartiments (chloroplaste, peroxysome, mitochondrie et cytosol). Sept des huit enzymes du cycle photorespiratoire sont phosphorylables. La phosphoglycolate phosphatase (AtPGLP1), première enzyme du cycle, est associée à quatre phosphosites. Des approches in vitro et in planta développées chez A. thaliana ont permis d’acquérir de nouvelles données sur la régulation post-traductionnelle de cette protéine, à la fois par phosphorylation et par oxydo-réductionAs sessile organisms, plants need to rapidly and effectively react to environmental abiotic and biotic stresses. To do so, various regulatory mechanisms exist that include post-translational modifications (PTMs) of proteins. One of the most prevalent PTM is protein phosphorylation that has been shown to occur in many metabolic pathways. Glycolysis allows the production of energy (as ATP) and reducing power from glucose. In this context, the regulation of Arabidopsis thaliana phosphoglycerate mutase (AtiPGAM) was studied by analysing a phosphorylation site potentially involved in the reaction mechanism of this glycolytic enzyme. The photorespiratory cycle is a major metabolic pathway occurring in all photosynthetic organisms. It is initiated by the oxygenase activity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and leads to the production of toxic 2-phosphoglycolate (2-PG) molecules. The costly recycling of 2-PG by the photorespiratory cycle takes place in four different compartments (chloroplast, peroxisome, mitochondrion and cytosol). Seven of the eight core photorespiratory enzymes appear to be phosphorylated. Phosphoglycolate phosphatase (AtPGLP1), the first enzyme of the cycle that metabolizes 2-PG to glycolate, is associated with four phosphosites. In vivo and in vitro approaches using Arabidopsis thaliana have allowed us to obtain further insights into the post-translational regulation of this protein by protein phosphorylation and by oxidation-reduction
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Murray, Jeremy Dale. "A genetic linkage study of obesity in a 3-generation Canadian kindred and investigation of the glycolytic regulatory enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a candidate gene." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/mq21097.pdf.
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Maluf, Fernando Vasconcelos. "Estudos estruturais e de química medicinal aplicados às enzimas da via glicolítica de protozoários: enolase de Plasmodium falciparum e gliceraldeído-3-fosfato desidrogenase de Trypanosoma cruzi." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-02102015-093453/.
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A melhor compreensão dos mecanismos fisiopatológicos e farmacológicos aliados a métodos modernos de investigação tornaram possível a descoberta e o desenvolvimento de fármacos para diversas doenças e disfunções orgânicas em humanos. Os fármacos desenvolvidos atualmente são resultados de intensos esforços em pesquisa por equipes multidisciplinares, impactando diretamente na qualidade de vida das diversas populações no mundo. Nesse cenário, os grupos de pesquisas estabelecidos em Universidades com foco no planejamento de fármacos para doenças tropicais têm crescido. A Malária e a Doença de Chagas figuram com especial importância, a primeira pela expressiva mortalidade mundial, enquanto a segunda pela morbidade e seus impactos na população brasileira. O tratamento de ambas possui limitações que se agravam, seja pelo baixo número de opções terapêuticas, ou pelo desenvolvimento de cepas resistentes. As enzimas investigadas nesse doutoramento, enolase (PfEnolase) de Plasmodium falciparum e gliceraldeído3fosfato desidrogenase de Trypanosoma cruzi (TcGAPDH), são componentes da via glicolítica destes parasitas e são considerados alvos moleculares atrativos para o desenvolvimento de inibidores enzimáticos, dada a importância destas enzimas no processo de obtenção de energia do parasita. Os estudos fundamentamse na busca por modulação seletiva da atividade biológica dos alvos selecionados através do desenvolvimento de novas moléculas bioativas. O estabelecimento de protocolo de expressão e purificação para enzima Pfenolase permitiu sua obtenção em quantidade e pureza suficiente para condução de estudos cinéticos e de triagem biológica, com a identificação de cinco novas classes químicas bastante promissoras; além de ensaios de cristalização, que culminaram na determinação da enzima em diversos complexos cristalográficos. Os dados estruturais produzidos foram fundamentais para condução da abordagem computacional de triagem virtual, que permitiu a identificação de 31 moléculas candidatas a inibidoras de Pfenolase. Avanços significativos foram obtidos também com a enzima TcGAPDH, destacando-se as adaptações nos processos de obtenção da proteína recombinante e ensaio cinético, condução de ensaio de bioprospecção orientada com a identificação e caracterização da molécula isolada (tilirosídeo). Novas condições de cristalização foram identificadas e poderão ser empregadas no processo de obtenção de complexos cristalográficos futuros. Adicionalmente, desenvolveu-se uma ferramenta computacional, Kinecteasy, para processamento automatizado dos dados produzidos das etapas de triagem biológica. Os trabalhos integrados de biologia estrutural e química medicinal desenvolvidos contribuem significativamente para o avanço no processo de planejamento de novos inibidores para as enzimas selecionadas.A better understanding of the pathophysiological and pharmacological mechanisms together with the modern research methods made possible the discovery and development of drugs for several humans´ diseases. The drugs currently developed are the result of intense efforts in research of multidisciplinary teams having as a direct consequence a remarkable impact on life quality of populations all over the world. In this scenario, research groups established at universities, with their focus on drug development for tropical diseases, are increasing. Malaria and Chagas disease deserve special attention, the former by the expressive world mortality, while the second by the morbidity and its impact on Brazilian population. Treatment for both has limitations, whether by the low number of therapeutic options, or by development of resistance. The target enzymes for this PhD project, enolase (PfEnolase) of Plasmodium falciparum and glyceraldehyde 3-phosphate dehydrogenase from Trypanosoma cruzi (TcGAPDH), are essential components of glycolytic pathway and therefore related to the parasite energy production, thus, are considered attractive molecular targets for enzyme inhibitors development. Essentially, the proposed studies seek selective modulation of the target´s biological activity through the development of new bioactive molecules. The expression and purification protocols developed for Pfenolase have allowed us to obtain recombinant protein at suitable yield and purity for conducting screening assays, which has revealed five new chemical classes as Pfenolase inhibitors. Crystallization experiments were successfully conducted and 3D structure were determined for different complexes. Structural data was essential for performing the computational approach of virtual screening, which has allowed us to identify 31 inhibitor candidates for Pfenolase. Significant advances were obtained with TcGAPDH, highlighting the adaptations on recombinant protein protocol and kinetic assay. Assay-guided bioprospecting experiments were successfully performed with identification and characterization of isolated inhibitor (tiliroside). New crystallization conditions were identified and will be employed in future co-crystallization and soaking studies. Additionally, Kinecteasy, a computational tool, were developed for automated data processing of biological screening assays. The structure and medicinal chemistry studies presented here contribute significantly in the process of drug development for the selected enzymes.
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Williams, Jonathan Glyn. "Isoenzyme specific PFK-2/FBPase-2 inhibition as an anti-cancer strategy." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:7f47d9bb-7a9d-4dbc-92fa-57d2654640d1.
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High aerobic glycolytic capacity is correlated with poor prognosis and increased tumour aggressiveness. 6Phosphofructo-1-kinase catalyses the first irreversible step of glycolysis, and is activated by fructose-2,6-bisphosphate, a product of the kinase activity of four bifunctional isoenzymes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFK-2/FBPase-2:PFKFB1-4). These are potential anti-tumour targets, but their individual and collective role requires further investigation. This thesis had three aims; to validate the PFK-2/FBPase-2 isoenzymes as anti-cancer targets, to investigate the requirement for isoenzyme-specific targeting, and to initiate assay development, enabling future identification of novel inhibitors. A panel of cancer cell lines was examined and PFKFB3 and PFKFB4 were confirmed to be the most strongly induced isoenzymes in hypoxia, regulated by HIF-1α. Basal and hypoxic relative PFKFB3/PFKFB4 expression varied markedly, and three cell lines with varying expression ratios (MCF-7, U87, PC3) were selected for further study. siRNA knockdown of each isoenzyme individually, markedly reduced 2D and 3D cell growth. The effect of PFKFB3 knockdown was consistently more pronounced, particularly in hypoxia. Double PFKFB3/PFKFB4 knockdown was significantly less effective than PFKFB3 knockdown alone. Direct antagonism of PFKFB3 and PFKFB4 on F-2,6-BP concentration was observed, with PFKFB3 exhibiting high kinase activity, as anticipated, and PFKFB4 exhibiting high bisphosphatase activity. The degree of antagonism was dependent on the relative PFKFB3/PFKFB4 expression ratio. Extensive efforts were made to examine the wider metabolic effect of PFKFB3/PFKFB4 on flux towards glycolysis or the pentose phosphate pathway (PPP), including using metabolite, lipid droplet, 13C NMR and mass spectrometry assays. No significant change in metabolic flux was detected, the evidence presented therefore suggesting the impact of the antagonistic effects of the isoenzymes on [F-2,6-BP] extends beyond regulation of metabolic flux alone. This study concluded that the most effective therapeutic strategy will be one that involves a PFKFB3-specific inhibitor, preferably hypoxia-targeted. Accordingly, steps were taken to validate and optimise a robust medium-throughput assay system.
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Abbaraju, Naga Vijayalaxmi. "Patterns of protein expression in tissues of the killifish, Fundulus heteroclitus and Fundulus grandis." ScholarWorks@UNO, 2011. http://scholarworks.uno.edu/td/113.
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Fundulus is a diverse and widespread genus of small teleost fish of North America. Due to its high tolerance for physiochemical variation (e.g. temperature, oxygen, salinity), Fundulus is a model organism to study physiological and molecular adaptations to environmental stress. The thesis focuses on patterns of protein expression in Fundulus heteroclitus and F. grandis.The patterns of protein expression were investigated using traditional methods of enzyme activity measurements and recent proteomic approaches. The findings of the study can be used to guide future studies on the proteomic responses of vertebrates to environmental stress. Chapter 2 focuses on measurement of the temporal effects of oxygen treatments on the maximal specific activities of nine glycolytic enzymes in liver and skeletal muscle during chronic exposure (28d) of Fundulus heteroclitus. The fish was exposed to four different oxygen treatments: hyperoxia, normoxia, moderate hypoxia, and severe hypoxia. The time course of changes in maximal glycolytic enzyme specific activities was assessed at 0, 8, 14 and 28 d. The results demonstrate that chronic hypoxia alters the capacity for carbohydrate metabolism in F. heteroclitus, with the important observation that the responses are both tissue- and enzyme-specific. Chapter 3 studies the effect of tissue storage on protein profile of tissues of F. grandis. The technique of one dimensional gel electrophoresis (1D-SDS-PAGE) was used to assess the effects of tissue sampling, flash frozen in liquid nitrogen versus immersion of fresh tissue in RNA later, for five tissues, liver, skeletal muscle, brain, gill, and heart, followed by LC-MS/MS to identify protein bands that were differentially stabilized in gill and liver. The study shows that, in F. grandis, the preferred method of preservation was tissue specific. xi Chapter 4 focuses on the use of advanced 2DE-MS/MS to characterize the proteome of multiple tissues in F. grandis. Database searching resulted in the identification of 253 non-redundant proteins in five tissues: liver, muscle, brain, gill, and heart. Identifications include enzymes of energy metabolism, heat shock proteins, and structural proteins. The protein identification rate was approximately 50 % of the protein spots analyzed. This identification rate for a species without a sequenced genome demonstrates the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.
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Metón, Teijeiro Isidoro. "Regulación nutricional de enzimas clave en la glucolisis-gluconeogénesis: expresión del gen 6-PF 2-K/FRU 2,6-P(2)ASA en hígado de Sparus aurata." Doctoral thesis, Universitat de Barcelona, 1996. http://hdl.handle.net/10803/671741.
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A través de estudios de ayuno y realimentación, y de tipo y cantidad de dieta suministrada, hemos comprobado cómo afecta el estado nutricional a enzimas clave implicadas en glucólisis-gluconeogénesis, vía de las pentosas fosfato y metabolismo de aminoácidos en hígado del pez teleósteo Sparus aurata. La investigacion se ha centrado en el control de la expresión génica de la enzima bifuncional 6-PF 2-K/FRU 2,6-P2ASA, responsable de la sintesis y degradación de FRU 2,6-P2, principal activador de la glucólisis e inhibidor de la gluconeogénesis. Flujo glucolítico, ruta de las pentosas, así como actividad quinasa, cantidad de proteína y niveles de mRNA de 6-PF 2-K/FRU 2,6-P2ASA, parámetros todos disminuídos por el ayuno, correlacionaron con la cantidad de dieta suministrada y con el nivel de carbohidratos aportado por la dieta. Esta enzima ha mostrado ser regulada a corto plazo fundamentalmente por alosterismo y/o modificación covalente, y a largo plazo por el nivel de proteína y mensajero. El análisis de los niveles de mRNA por Northern Blot se ha realizado con una sonda homóloga obtenida por RT-PCR, cuya secuencia es la primera descrita para el mensajero de 6-PF 2-K/FRU, 2,6-P2ASA en peces.
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Burger, Vincent. "Substrats suicide potentiels d'enzymes : synthese et resultats biologiques." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13140.
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Menard, Guillaume. "Recherche d'haplotypes enzymatiques associés à des phénotypes métaboliques chez la tomate." Thesis, Bordeaux 1, 2012. http://www.theses.fr/2012BOR14648/document.
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La recherche de variations d’activités enzymatiques associées à des phénotypes chez la tomate (Solanum Lycopersicum) a permis d’apporter de nouveaux éléments pour l’étude desrelations existantes entre le métabolisme central et la qualité du fruit. Ce projet qui engageaitune nouvelle thématique au sein de l’unité Biologie et Pathologie de Fruit (UMR 1332, INRABORDEAUX) a permis dans un premier temps de développer une plateforme d’enzymologieà haut débit. Cette structure permet d’une part de réaliser plus de 10 000 déterminationsd’activités enzymatiques par jour avec une très grande reproductibilité et d’autre part dedéterminer les constantes de Michaelis (Km) apparentes pour jusqu’à une dizaine d’enzymespar jour.Dans un deuxième temps ce projet s’est attaché à étudier les relations existantes entre lesenzymes de la voie de la glycolyse chez le cultivar de tomate MicroTom. Nous y avonsrelevé l’existence de corrélations fortes entre les activités de ces enzymes. Nous avonségalement mis à jour l’existence de corrélations pour les enzymes mesurées à partir de deuxétages foliaires distincts ce qui suggère que les réseaux enzymatiques sont conservés ausein d’une plante.Dans un troisième temps, le criblage d’une collection de mutants de tomate MicroTom a étéentrepris. Ce criblage de plus de 150 familles (soit environ 1800 plantes) sur la base desactivités enzymatique de onze enzymes du métabolisme central à deux concentrations desubstrats différentes (saturante et non saturante) a abouti à l’identification de deux familles.Ces deux familles porteraient chacune une mutation affectant les caractéristiques cinétiquesde la Triose-Phosphate Isomérase. Ces mutations étaient toujours en cours d’étude à la finde ce projet. Ces résultats ouvrent de nouvelles perspectives pour la compréhension desrelations entre les enzymes du métabolisme central. Ils permettent aussi d’apporter desméthodes d’identification rapide de mutants enzymatiques au sein d’une large populationResearch on enzymatic variations associate with phenotypes in tomato (SolanumLycopersicun) provided new and original input regarding links between central metabolismand fruit quality. This project took part in a new topic of the Fruit Biology and Pathology Unit(UMR 1332, INRA BORDEAUX). First, during this project, a new high-throughputenzymology platform was created. This unique lab offers possibility to determine both morethan 10 000 enzyme activities per day with a very good reliability and apparent Michealisconstant (Km) for up to ten enzymes per day.Second, this project investigated existent relationship between glycolytic enzymes inMicroTom tomato cultivar. We highlighted strong correlations between enzymes in leaves.We also uncovered correlations between enzymes activities measured in two distinct foliarlevels. These elements suggest inheritability of the enzymes network within the plant.Third, the screen of an Ethylmethyl Sulfonate (EMS) MicroTom mutant’s collection wasinitiated. 150 families (around 1800 plants) were screened for eleven enzymes with twodifferent substrate concentrations. At the end of the process, two families were identified;both could have mutation(s) that affect(s) the kinetic characteristic of Triose-Phosphateisomerase. These mutations were style investigated at the end of this project. These originalresults provide new perspectives for knowledge of relationship between central metabolism’senzymes. Finally, This project proposes new and rapid enzymatic mutant identification withina large population as an EMS mutant’s collection
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Connolly, Siobhan. "Etude biochimique et physicochimique de gommes végétales exsudées par Acacia senegal et Combretum nigricans." Rouen, 1988. http://www.theses.fr/1988ROUES005.
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Betbeder, Didier. "Synthese et etude du mode d'action d'inhibiteurs de voies metaboliques du trypanosome." Toulouse 3, 1988. http://www.theses.fr/1988TOU30029.
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Etude d'inhibiteurs de voies metaboliques du trypanosome. Synthese et action d'inhibiteurs possibles d'enzymes de replication. Etude d'enzymes de la glycolyse, cible cle dans le trypanosome, celui-ci utilisant le glucose comme seule source d'energie. Puis caracterisation d'une proteine qui presente les proprietes de recepteur vis-a-vis d'une nouvelle famille de trypanocides derives du diphenyl methane
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Wojtera, Joanna. "Microcompartmentation of plant glycolytic enzymes with subcellular structures." Doctoral thesis, 2009. https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2009102118.
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Classically considered as a soluble system of enzymes, glycolysis does not conform to the known function and subcellular microcompartmentation pattern. Certain glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be found at different cellular locations in animal cells, where it exhibited its non-glycolytic activities. Determination of the subcellular localization of two cytosolic GAPDH isoforms from Arabidopsis thaliana (GapC1 and GapC2), fused to Fluorescent Proteins (FP), was investigated in the transiently transformed mesophyll protoplasts, using confocal Laser Scanning Microscopy. Apart from its cytosolic distribution, the nuclear compartmentation of GapC:FP was observed in this study, as well as its punctuate or aggregate-like localization. Part of the GapC:FP foci were observed as mitochondria-associated. A further yeast two-hybrid screen with both GapC isoforms as baits allowed to identify the mitochondrial porin (VDAC3; At5g15090) as a protein-protein interaction partner. Further tests with one-on-one yeast two-hybrid and Bimolecular Fluorescence Complementation (BiFC) assays showed that the detected binding between plant VDAC3 and GapC in yeast cells was false positive. Interestingly, aldolase interacted with VDAC3, as well as with GapC in plant protoplasts, using the BiFC method. On the other hand, no such interaction could be detected in the one-on-one yeast two-hybrid assay. Thus, a model of indirect mitochondrial association of GapC via aldolase that binds directly to mitochondrial porin is proposed to occur only upon certain cellular conditions. Attempts to show the binding of Arabidopsis GAPDH to the actin cytoskeleton in vivo failed, whereas in vitro cosedimentation assays demonstrated that the fully active, recombinant glycolytic enzyme binds to rabbit F-actin. Moreover, is the presence of the GapC cofactor NAD and a reducing agent (DTT), the enzyme might exhibit an actin-bundling activity.
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Wanty, Christopher James. "Directed evolution of glycolytic enzymes : glyceraldehyde-3-phosphate dehydrogenases." Phd thesis, 2010. http://hdl.handle.net/1885/151459.
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Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) performs a vital step in glycolysis. An in vitro fermentative system is being developed to convert glucose to ethanol. The system is not cofactor neutral, so will eventually stall through production of adenosine triphosphate (ATP). The Gapdh catalysed step can be modified by use of a non-phosphorylating variant (GapN) of the enzyme. GapN catalyses the Gapdh oxidation and the next step of the pathway without the production of ATP through incorporation of phosphate that normally occurs. This results in the pathway producing ethanol and carbon dioxide from glucose, with complete cofactor recycling. This also gives an overall thermodynamic advantage to the pathway. A GapN enzyme from the thermophile Thermoproteus tenax was examined for suitability to this role, and was modified to increase activity under mesophilic conditions through application of directed evolution techniques. Methods for evolving this enzyme were developed and are described. Two mutants from the evolution are characterised. Both possess a threefold enhanced activity at 30{u00B0}C and one and a half to tenfold improvement at 50{u00B0}C. One suggests a change in optimal active temperature, while the other has improved activity under all conditions tested. The first heat activation of GapN was observed in both the wild type and the mutants, with a sevenfold improvement in wild-type activity and a fourfold and modest improvement in the mutants. This phenomenon is not commonly reported outside of heat-response proteins. Alternative screening methods were investigated for the evolution. Primarily an E. coli strain lacking the gene gapA, that encodes Gapdh, was to be used for screening. There were significant difficulties knocking out gapA that are now believed to be caused by non-glycolytic roles of Gapdh in the cell. A second evolution was performed with a mutant of the E. coli Gapdh possessing a cysteine 149 to alanine mutation. This mutation causes a GapN like activity in Gapdh, though it is understood to occur by a different mechanism and to use a different isomer of the substrate for activity. Following two rounds of evolution an enzyme with a wild-type cysteine at position 149 was selected. This suggests potential problems with the C149A mutant as an evolution target. A third Gapdh variant from Zymomonas mobilis was investigated for possible use in the pathway, or as a possible target for evolution. This variant has not been previously characterised. The first kinetic characterisation and crystal structures are reported. The enzyme is kinetically interesting for its strong substrate binding of all substrates with Km at low micromolar ranges and a turnover of 16 s-1 that could be attributed to the environment of Z. mobilis. The best crystallographic data collected have given a structural solution to 3.6 A.
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Wojtera, Joanna [Verfasser]. "Microcompartmentation of plant glycolytic enzymes with subcellular structures / vorgelegt von Joanna Wojtera." 2009. http://d-nb.info/99758095X/34.
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Li, Wei. "Regulation of the synthesis of key gluconeogenic and glycolytic enzymes by biotin." 1992. http://hdl.handle.net/1993/18537.
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Yu, Chen-Chiao, and 游甄巧. "Hypoxic effects on early stages of zebrafish embryogenic development and expression of glycolytic enzymes." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/57768901053395270796.
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碩士國立臺灣海洋大學
生命科學暨生物科技學系
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During the early stage of development, the energy metabolic pathway in the rapidly growing and dividing embryonic cells is different from that in mature cells. In mammals, the proliferating embryonic cells obtain energy through glycolysis. The metabolic products of glycolysis also support the requirements in protein, lipid and nucleic acid biosynthesis in the proliferating cells. The hypoxia-inducible factors, including HIF1α and HIF2α, play critical roles in neural and blood cell development in zebrafish embryos, suggesting that these factors are present in active form in the developing embryos to regulate various downstream genes. This study aims to investigate the expression pattern of glycolytic enzymes during development and the relationship between the HIF proteins and glycolytic genes as well as their responses to hypoxia stress. It was shown that the transcripts of glycolytic genes can be detected at 1-cell stage, indicating the transcripts of these genes are stored as maternal mRNA. The encoded mRNA of these glycolytic genes is widely distributed in whole embryos without specificity before the segmentation stage. After then, these genes are expressed with tissue specific pattern. Although hypoxic exposure did not affect the glycolytic genes during early stages of development, temporal or consistent hypoxia treatment delayed embryonic development. Depletion of HIF caused more severe defects in those hypoxia-exposed embryos, suggesting the HIF-related pathways play important functions in protecting embryos against hypoxic stress. Nevertheless, these protections are not mediated by enhancing the expression of glycolytic genes. It is unclear how hypoxic stress affects glycolytic genes in the embryos and delays development. This is the first time to show that hypoxia does not affect glycolytic genes during early stages of development and the HIF-related pathways protect embryos against hypoxia-induced developmental delays. Keywords: Hypoxia, glycolysis, growth delay, HIF1α, HIF2α, HIF3α
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Banerjee, Mousumi. "Structure-Function Studies On Triosephoshate Isomerase From Plasmodium falciparum And Methanocaldococcus jannaschii." Thesis, 2008. http://hdl.handle.net/2005/824.
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This thesis describes studies directed towards understanding structure-function relationships of triosephosphate isomerase (TIM), from a protozoan parasite Plasmodium falciparum and a thermophilic archaea Methanocaldococcus jannaschii. Triosephosphate isomerase, a ubiquitous glycolytic enzyme, has been the subject of biochemical, enzymatic and structural studies for the last five decades. Studies on TIM have been central to the development of mechanistic enzymology. The present study investigates the role of specific residues in the structure and function of Plasmodium falciparum triosephosphate isomerase (PfTIM). The structure and stability of a tetrameric triosephosphate isomerase from Methanocaldococcus jannaschii (MjTIM) is also presented.
Chapter 1 provides a general introduction to the glycolytic enzyme triosephosphate isomerase, conservation of TIM sequences, its fold and three dimensional organization. The isomerisation reaction interconverting dihydroxyacetone phosphate and glyceraldehyde 3phosphate catalyzed by triosephosphate isomerase is an example of a highly stereospecific proton transfer process (Hall & Knowles, 1975; Rieder & Rose, 1959). This chapter briefly reviews mechanistic features and discusses the role of active site residues and the functional flexible loop 6. Triosephosphate isomerase adopts the widely occurring ( β/ α)8 barrel fold and mostly occurs as a dimer (Banner et al., 1975). Protein engineering studies, related to folding, stability and design of monomeric TIM are also addressed. A brief introduction to thermophilic TIMs and higher oligomeric TIMs is given. The role of this enzyme in disease states like hemolytic anemia and neuromuscular dysfunction is surveyed. The production of methylglyoxal, a toxic metabolite, as a byproduct of the TIM reaction is also considered.
Many proteins utilize segmental motions to catalyze a specific reaction. The omega loop (loop 6) of triosephosphate isomerase is important for preventing the ene-diol intermediate from forming the cytotoxic byproduct, methylglyoxal. The active site loop-6 of triosephosphate isomerase moves about 7Ǻ on ligand binding. It exhibits a hinged lid motion alternating between two well defined, “open” and “closed”, conformations (Joseph et al., 1990). Though the movement of loop 6 is not ligand gated, in crystals the ligand bound forms invariably reveal a closed loop conformation. Plasmodium falciparum TIM is an exception which predominantly exhibits “open” loop conformations, even in the ligand bound state (Parthasarathy et al., 2002). Phe 96 is a key residue that is involved in contacts between the flexible loop-6 and the protein body in PfTIM. Notably, in all TIM sequences determined thus far, with the exception of plasmodial sequences, this residue is Ser 96. In Chapter 2 the mutants F96S, F96H and F96W are reported. The crystal structures of the mutant enzymes with or without bound ligand are described. In all the ligand free cases, loop-6 adopts an “open” conformation. Kinetic parameters for all the mutants establish that residue 96 does not play an essential role in modulating the loop conformation but may be important for ligand binding. Structural analysis of the mutants along with WT enzyme reveals the presence of a water network which can modulate ligand binding.
Subunit interfaces of oligomeric proteins provide an opportunity to understand protein- protein interactions. Chapter 3 describes biochemical and biophysical studies on two separate dimer-interface destabilizing mutants C13E and W11F/W168F/Y74W of PfTIM. The intention was to generate a stable monomer by disrupting the interaction hubs. C13 is a part of a large hydrophobic patch (Maithal et al., 2002a) at the dimer interface. Introduction of a negative charge at position 13 destabilizes the interface and reduces activity. Y74 is a part of an aromatic cluster of the interface (Maithal et al., 2002b). The Y74W triple mutant was designed to disrupt the aromatic cluster by introducing additional atoms. Tryptophan is also a fluorophore, allowing studies of the dimer disruption by fluorescence, after mutating the two inherent tryptophan residues, W11 and W168 to phenylalanine. The mutants showed reduced activity and were more sensitive than the wild type enzyme to chemical denaturants as well as thermal denaturation. Evidenced for monomer formation is presented. These studies together with previous work reveal that the interface is important for both activity and stability.
In order to develop a model for understanding the relationship between protein stabilization and oligomeric status, characterization of the TIM from Methanocaldococcus jannaschii (MjTIM) has been undertaken. Chapter 4 describes the purification and characterization of MjTIM. The MjTIM gene was cloned and expressed in pTrc99A and protein was isolated from AA200 E. coli cells. Hyperexpressed protein was purified to homogeneity and relevant kinetic parameters have been determined. The tetrameric nature of MjTIM is established by gel filtration studies. Circular dichroism (CD) studies establish the stability of the overall fold, even at temperatures as high as 95ºC. A surprising loss of enzyme activity upon prolonged incubation at high temperature was observed. ESI-MS studies establish that oxidation of thiol groups of the protein may be responsible for the thermal inactivation.
Chapter 5 describes the molecular structure of MjTIM, determined in collaboration with Prof. MRN Murthy’s group at the Indian Institute of Science (Gayathri et al., 2007). The crystal structure of the recombinant triosephosphate isomerase (TIM) from the archaeabacteria Methanocaldococcus jannaschii has been determined at a resolution of 2.3 Å. MjTIM is tetrameric, as suggested by solution studies and from the crystal structure, as in the case of two other structurally characterised archaeal TIMs. The archaeabacterial TIMs are shorter compared to the dimeric TIMs, with the insertions in the dimeric TIMs occurring in the vicinity of the putative tetramer interface, resulting in a hindrance to tetramerization in the dimeric TIMs. The charge distribution on the surface of archaeal TIMs also facilitates tetramerization. Analysis of the barrel interactions in TIMs suggests that these interactions are unlikely to account for the thermal stability of archaeal TIMs. A feature of the unliganded structure of MjTIM is the complete absence of electron density for the loop 6 residues. The disorder of the loop may be ascribed to a missing salt bridge between residues at the N- and C- terminal ends of the loop in MjTIM.
Chapter 6 is a follow up of an interesting observation made by Vogel and Chmielewski (1994), who noticed that subtilisin cleaved rabbit muscle triosephosphate isomerase religated spontaneously upon addition of organic solvents. Further extension of this nicking and religation process with PfTIM emphasizes the importance of tertiary interactions in contributing to the stability of the (β/α)8 barrel folds (Ray et al., 1999). This chapter establishes that subtilisin nicking and religation is also facile in thermophilic MjTIM. Fragments generated by subtilisin nicking were identified using MALDI mass spectrometry at early and late stages of the cleavage for both the dimeric PfTIM and tetrameric MjTIM. This chapter also describes the comparative thermal and denaturant stability of both the enzymes. The accessibility of the Cys residues of MjTIM has been probed by examining the rates of labeling of thiol groups by iodoacetamide. The differential labeling of Cys residues has been demonstrated by mass spectrometry.
Chapter 7 summarizes the main results and conclusions of the studies described in this thesis.