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Ekici, Özlem Doğan. "Design, synthesis, and evaluation of novel irreversible inhibitors for caspases." Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04062004-164633/unrestricted/ekici%5Fozlem%5Fd%5F200312%5Fphd.pdf.
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Qian, Yuhui. "Study of Basic Wood Decay Mechanisms and Their Biotechnological Applications." Fogler Library, University of Maine, 2008. http://www.library.umaine.edu/theses/pdf/QianY2008.pdf.
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Moore, Robert Goodwin Douglas C. "Towards the understanding of complex biochemical systems the significance of global protein structure and thorough parametric analysis /." Auburn, Ala, 2009. http://hdl.handle.net/10415/1766.
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Ekici, Ozlem Dogan. "Design, synthesis, and evaluation of novel irreversible inhibitors for caspases." Diss., Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/5333.
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Epstein, Todd Matthew. "Structural and kinetic studies of two enzymes catalyzing phospholipase A2 activity." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 2.39 Mb., 186 p, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3200538.
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Fisher, Oriana. "Subcloning, enzymatic characterization, and in silico docking of transglutaminase 2." Waltham, Mass. : Brandeis University, 2009. http://dcoll.brandeis.edu/handle/10192/23253.
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Åström, Nina. "NADH/NAD⁺ analogues and cyclodextrins in enzyme mimicking systems an experimental and computational investigation /." Lund : Organic Chemistry 1, Lund University, 1995. http://catalog.hathitrust.org/api/volumes/oclc/39781586.html.
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Baloyi, Thembekile Feonah. "Effects of exogenous fibrolytic enzymes on in vitro fermentation kinetics of forage and mixed." Thesis, Stellenbosch : Stellenbosch University, 2008. http://hdl.handle.net/10019.1/19895.
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Thesis (MScAgric)--Stellenbosch University, 2008.ENGLISH ABSTRACT: Two in vitro experiments were conducted to evaluate the effect of exogenous fibrolytic enzyme application on dry matter (DM) and neutral detergent fibre (NDF) degradation and gas production (GP) of mature forages and forage-concentrate mixtures. The forages used in the first experiment were lucerne hay (LH), oat hay (OH) and wheat straw (WS). The same forages were used in the second experiment, but they were mixed with a concentrate feed to make three mixtures consisting of 80% (HC), 50% (MC) or 20% (LC) concentrate. The extracellular enzyme fraction (supernatant) of a fungal strain, ABO 374, was used as feed additive. The supernatant was used in a fresh (SU-ABO374) or lyophilized (CSIR-ABO374) form, the latter being reconstituted with water immediately before application. The liquid supernatants were applied to the incubation medium and not directly to the substrate, at a rate equivalent to 7.5 ml/kg feed DM. In the control treatments of both experiments, water was used instead of the liquid supernatants. For the DM and NDF degradability trials in both experiments, 500 mg forage samples were weighed into 50 x 50 mm dacron bags which were incubated anaerobically at 39ºC in 1.4L of a rumen liquid inoculated buffered medium in 2L fermentation jars. Bags from all treatments were removed after 2, 4, 8, 12, 24, 48, 72 and 96 h of incubation. For the gas production determinations, 500 mg of the respective substrate samples were weighed into 120 ml glass vials which were incubated for 96 h in 40 ml inoculated medium to which 0.5 ml of the respective enzyme solutions were added. Gas pressure was recorded manually with a digital pressure gauge after 2, 4, 8, 12, 24, 48, 72 and 96 h and pressure was converted to volume with a predetermined regression. The 96 h substrate residues were washed, dried, weighed and analyzed for NDF and OM. In both experiments the substrates differed in terms of DM and NDF degradability and gas production rates, but the enzyme treatments had no effect. The lack of response to enzyme application was ascribed to a number of factors, including the fact that enzyme application was into the incubation medium and not directly onto the substrates and also that no significant pre-incubation interaction time was allowed. The same preparations gave positive results in previous trials where they were applied directly onto the substrates and where a pre-incubation interaction time of 16 hours was allowed. (Key words: Exogenous enzymes, forages, concentrate based diets, DM and NDF degradation, gas production )
AFRIKAANSE OPSOMMING: Die invloed van eksogene fibrolitiese ensieme op in vitro fermentasiekinetika van ruvoer- en gemengde voersubstrate. Twee in vitro-experimente is uitgevoer om die invloed van eksogene fibrolitiese ensieme op droëmateriaal (DM) en neutraal-onoplosbare vesel (NDF) degradering en gasproduksie (GP) van volwasse ruvoersubstrate en ruvoer-kragvoermengsels te bepaal. Ruvoere in die eerste eksperiment was lusernhooi (LH), hawerhooi (HH) en koringstrooi (KS). Dieselfde ruvoere is in die tweede eksperiment gebruik, maar hulle is met ‘n kragvoer gemeng om drie mengsels te maak, bestaande uit 80% (HK), 50% (MK) of 20% (LK) kragvoer. Die ekstrasellulêre ensiemfraksie (supernatant) van ‘n fungiale stam, ABO 374, is as ‘n voertoedieningsmiddel gebruik. Die supernatant is is in ‘n vars (SU-ABO374) of gevriesdroogde (WNNR-ABO374) vorm gebruik, waar laasgenoemde onmiddellik voor toediening gerekonstitueer is. Die vloeistof-supernatante is nie direk op die substrate gevoeg nie, maar tot die inkubasiemedium gevoeg, teen ‘n hoeveelheid ekwivalent aan 7.5 ml/kg voer DM. In die kontrolebehandeling van beide eksperimente, is water in plaas van die vloeistofsupernatante gebruik. Vir die DM- en NDF-degraderingsproewe in beide eksperimente, is 500 mg van die onderskeie ruvoere in 50 x 50 mm dacronsakkies geweeg wat anaerobies by 39ºC geïnkubeer is in 1.4L van ‘n rumenvloeistof-geïnokkuleerde medium in 2L fermentasieflesse. Vir alle behandelings is sakkies na 2, 4, 8, 12, 24, 48, 72 en 96 h inkubasie verwyder. Vir gasproduksiebepalings is 500 mg van die onderskeie substraatmonsters in 120 ml glasbotteltjies geweeg en vir 96 h in 40 ml geïnokkuleerde medium geïnkubeer waarin 0.5 ml van die onderskeie ensiemoplossings gevoeg is. Gasdruk is na 2, 4, 8, 12, 24, 48, 72 en 96 h bepaal met behulp van ‘n digitale drukmeter en druk is met behulp van ‘n voorafbepaalde regressie na volume omgeskakel. Die 96 h substraatresidue is gewas, gedroog, geweeg en ontleed vir NDF en OM. In beide eksperimente het die substrate verskil ten opsigte van DM- en NDF-degradeerbaarheid en gasproduksietempo’s, maar die ensiembehandelings het geen invloed gehad nie. Die gebrek aan respons is aan verskeie faktore toegeskryf, insluitend die feit dat ensiemtoediening in die inkubasiemedium toegedien is en nie direk op die substrate nie, asook die feit dat daar nie ‘n noemenswaardige pre-inkubasie interaksietyd toegalaat is nie. Dieselfde ensiempreparate het positiewe resultate gelewer in vorige proewe waar dit direk op die substraat toegedien is en waar ‘n pre-inkubasie interaksietyd van 16 ure toegelaat is. (Sleutelwoorde: Eksogene ensieme, ruvoere, kragvoerdiëte, DM- en NDF-degradering, gasproduksie)
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Finnigan, William John Andrew. "The exploitation of thermophiles and their enzymes for the construction of multistep enzyme reactions from characterised enzyme parts." Thesis, University of Exeter, 2016. http://hdl.handle.net/10871/27323.
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Biocatalysis is a field rapidly expanding to meet a demand for green and sustainable chemical processes. As the use of enzymes for synthetic chemistry becomes more common, the construction of multistep enzyme reactions is likely to become more prominent providing excellent cost and productivity benefits. However, the design and optimisation of multistep reactions can be challenging. An enzyme toolbox of well-characterised enzyme parts is critical for the design of novel multistep reactions. Furthermore, while whole-cell biocatalysis offers an excellent platform for multistep reactions, we are limited to the use of mesophilic host organisms such as Escherichia coli. The development of a thermophilic host organism would offer a powerful tool allowing whole-cell biocatalysis at elevated temperatures. This study aimed to investigate the construction of a multistep enzyme reaction from well-characterised enzyme parts, consisting of an esterase, a carboxylic acid reductase and an alcohol dehydrogenase. A novel thermostable esterase Af-Est2 was characterised both biochemically and structurally. The enzyme shows exceptional stability making it attractive for industrial biocatalysis, and features what is likely a structural or regulatory CoA molecule tightly bound near the active site. Five carboxylic acid reductases (CARs) taken from across the known CAR family were thoroughly characterised. Kinetic analysis of these enzymes with various substrates shows they have a broad but similar substrate specificity and that electron rich acids are favoured. The characterisation of these CARs seeks to provide specifications for their use as a biocatalyst. The use of isolated enzymes was investigated as an alternative to whole-cell biocatalysis for the multistep reaction. Additional enzymes for the regeneration of cofactors and removal of by-products were included, resulting in a seven enzyme reaction. Using characterised enzyme parts, a mechanistic mathematical model was constructed to aid in the understanding and optimisation of the reaction, demonstrating the power of this approach. Thermus thermophilus was identified as a promising candidate for use as a thermophilic host organism for whole-cell biocatalysis. Synthetic biology parts including a BioBricks vector, custom ribosome binding sites and characterised promoters were developed for this purpose. The expression of enzymes to complete the multistep enzyme reaction in T. thermophilus was successful, but native T. thermophilus enzymes prevented the biotransformation from being completed. In summary, this work makes a number of contributions to the enzyme toolbox of well-characterised enzymes, and investigates their combination into a multistep enzyme reaction both in vitro and in vivo using a novel thermophilic host organism.
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Väljamäe, Priit. "The kinetics of cellulose enzymatic hydrolysis : Implications of the synergism between enzymes." Doctoral thesis, Uppsala University, Department of Biochemistry, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3120.
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The hydrolysis kinetics of bacterial cellulose and its derivatives by Trichoderma reesei cellulases was studied. The cellulose surface erosion model was introduced to explain the gradual and strong retardation of the rate of enzymatic hydrolysis of cellulose. This model identifies the decrease in apparent processivity of cellobiohydrolases during the hydrolysis as a major contributor to the decreased rates. Both enzyme-related (non-productive binding) and substrate-related (erosion of cellulose surface) processes contribute to the decrease in apparent processivity. Furthermore, the surface erosion model allows, in addition to conventional endo-exo synergism, the possibility for different modes of synergistic action between cellulases. The second mode of synergism operates in parallel with the conventional one and was found to be predominant in the hydrolysis of more crystalline celluloses and also in the synergistic action of two cellobiohydrolases.
A mechanism of substrate inhibition in synergistic hydrolysis of bacterial cellulose was proposed whereby the inhibition is a result of surface dilution of reaction components (bound cellobiohydrolase and cellulose chain ends) at lower enzyme-to-substrate ratios.
The inhibition of cellulases by the hydrolysis product, cellobiose, was found to be strongly dependent on the nature of the substrate. The hydrolysis of a low molecular weight model substrate, such as para-nitrophenyl cellobioside, by cellobiohydrolase I is strongly inhibited by cellobiose with a competitive inhibition constant around 20 μM, whereas the hydrolysis of cellulose is more resistant to inhibition with an apparent inhibition constant around 1.5 mM for cellobiose.
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Williams, Simon-Peter. "Studies of enzyme kinetics and aspects of enzyme structure in vivo using NMR and molecular genetics." Thesis, University of Oxford, 1992. http://ora.ox.ac.uk/objects/uuid:d8baa574-a5d4-45a2-95a2-c141fbf8d277.
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A quantitative understanding of metabolic control depends on a knowledge of the enzymes involved. The extrapolation of studies in vitro to the intact cell is controversial because the intracellular environment is relatively poorly characterised, particularly with respect to the interactions between weakly-associated enzymes. There is a clear need to study enzymes directly in the cell, yet there are few suitable techniques. Metabolites have been very successfully studied in cells by the non-invasive technique of nuclear magnetic resonance (NMR). NMR studies of enzymes in the cell have, however, been prevented by difficulties in assigning the resonances from the many proteins within the cell. A method for studying a specific enzyme in the cell has been developed, using Saccharomyces cerevisiae and phosphoglycerate kinase (PGK) as a model system. Using an inducible expression system, PGK was synthesised in the cell without significant synthesis of other proteins. With 5-fluorotryptophan in the growth medium, fluorine-labelled PGK was formed in situ. Fluorine is an excellent label for NMR since it is absent from most cells and has a high receptivity to NMR detection. 19 F NMR was used to study PGK in the intact cell. Comparisons with measurements in vitro showed that PGK was exposed to only a small fraction of the total intracellular [ADP], implying some form of compartmentalisation. The NMR relaxation properties observed in vivo and in vitro were compared with theoretical predictions. This showed that PGK was not part of a complex in the cell and that the viscosity of the cytoplasm, relative to water, was c. 4 at 30 °C. Fluorine-labelled pyruvate kinase and hexokinase have also been prepared; the spectra of these proteins in vitro are responsive to their ligands, and further work will study these proteins in vivo. NMR techniques were also applied to study the kinetics of PGK in the cell. PGK and GAPDH catalyse an ATP↔Pi exchange which is near-equilibrium in wild-type cells. 31P magnetisation transfer experiments in genetically manipulated cells showed that the reaction becomes unidirectional if the PGK activity is reduced by 95 %. Net flux is reduced by less than 30 %. In low-PGK cells, the ATP↔Pi exchange from oxidative phosphorylation can be isolated from that of glycolysis, facilitating direct measurements of the P:O ratio. In the cells studied, the P:O ratio was 2 to 3.
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Loftus, Katherine Marie. "Studies of the Structure and Function of E.coli Aspartate Transcarbamoylase." Thesis, Boston College, 2006. http://hdl.handle.net/2345/580.
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Thesis advisor: Evan R. KantrowitzE.coli Aspartate transcarbamoylase (ATCase) is the allosteric enzyme that catalyzes the committed step of the de novo pyrimidine biosynthesis pathway. ATCase facilitates the reaction between L-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate. The holoenzyme is a dodecamer, consisting of two trimers of catalytic chains, and three dimers of regulatory chains. ATCase is regulated homotropically by its substrates, and heterotropically by the nucleotides ATP, CTP, and UTP. These nucleotides bind to the regulatory chains, and alter the activity of the enzyme at the catalytic site. ATP activates the rate of ATCase's reaction, while CTP inhibits it. Additionally, UTP and CTP act together to inhibit the enzyme synergistically, each nucleotide enhancing the inhibitory effects of the other. Two classes of CTP binding sites have been observed, one class with a high affinity for CTP, and one with a low affinity. It has been theorized that the asymmetry of the binding sites is intrinsic to each of the three regulatory dimers. It has been hypothesized that the second observed class of CTP binding sites, are actually sites intended for UTP. To test this hypothesis, and to gain more information about heterotropic regulation of ATCase and signal transmission in allosteric enzymes, the construction of a hybrid regulatory dimer was proposed. In the successfully constructed hybrid, each of the three regulatory dimers in ATCase would contain one regulatory chain with compromised nucleotide binding. This project reports several attempts at constructing the proposed hybrid, but ultimately the hybrid enzyme was not attained. This project also reports preliminary work on the characterization of the catalytic chain mutant D141A. This residue is conserved in ATCase over a wide array of species, and thus was mutated in order to ascertain its significance
Thesis (BS) — Boston College, 2006
Submitted to: Boston College. College of Arts and Sciences
Discipline: Chemistry
Discipline: College Honors Program
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Geddes, Alexander. "A study of H-transfer kinetics and catalytic protein dynamics in ene-reductase enzymes of the OYE family." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/a-study-of-htransfer-kinetics-and-catalytic-protein-dynamics-in-enereductase-enzymesof-the-oye-family(b9a8338b-7917-4197-9870-261d90228495).html.
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Dynamic structural fluctuations occurring over a broad range of timescales are now known to facilitate the catalytic function of enzymes, but there is less comprehensive experimental evidence linking fast-timescale, high frequency motions to the reaction coordinate. Interest in the role of such motions has recently surged and been the subject of intensive experimental efforts, in part due to the identification of enzymatic hydride tunnelling reactions. This mechanism involves transiently degenerate product and reactant states, which enable H-transfer to occur instantaneously without the need to surmount the activation barrier associated with traditional transition-state based models of enzyme catalysis. The primary gauge of tunnelling in enzyme-catalysed reactions is the identification of temperature dependent kinetic isotope effects (KIEs), i.e. the relative rates of a reaction where the transferred atom is substituted for an alternate isotope. The identification of temperature-, and also pressure-, dependent KIEs has resulted in the emergence of new models of describing enzymatic H-transfer. These invoke a role for fast-timescale protein motions that 'promote' transfer via tunnelling. A popular model system for studying enzymatic H-tunnelling reactions is Pentaerythritol tetranitrate reductase, which belongs to the Old Yellow Enzyme (OYE) family of ene-reductases. These nicotinamide coenzyme dependent oxidoreductases catalyse the stereospecific reduction of alpha/β-unsaturated alkene containing substrates. Here, the importance of donor-acceptor distances in determining the observed rate of PETNR reduction with NAD(P)H is probed via a detailed structural and kinetic analysis of site-directed variants. In addition, an investigation of distance-dependent Nuclear Overhauser effects via Nuclear Magnetic Resonance (NMR) spectroscopy is undertaken to assess active site organisation and measure donor-acceptor distances in PETNR-substrate complexes. A variable pressure NMR study reveals how NOE build- up is perturbed in high-energy conformers favoured as a result of the application of increased hydrostatic pressures. Recently there has been interest in exploiting the stereoselective properties of reactions catalysed by ene-reductase enzymes for use in biocatalytic reactions to produce industrially valuable compounds from renewable sources. The reactions of PETNR and additional OYE enzymes, Thermophilic old yellow enzyme and Xenobiotic reductase A, with both natural coenzymes and a set of synthetic Nicotinamide Coenzyme Biomimetics (NCBs) are also characterised. The NCBs represent affordable and fast-reacting alternatives to the physiological coenzymes. Reactions with NCBS are also shown to proceed via a tunnelling mechanism and furthermore, that enhanced donor-acceptor sampling correlates with the faster reactivity seen with these compounds.
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Lee, Charles Kai-Wu. "Eurythermalism of a deep-sea symbiosis system from an enzymological aspect." The University of Waikato, 2007. http://hdl.handle.net/10289/2588.
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The recently proposed and experimentally validated Equilibrium Model provides the most detailed description of temperature's effect on enzyme catalytic activity to date. By introducing an equilibrium between Eact, the active form of enzyme, and Einact, a reversibly inactivated form of enzyme, the Equilibrium Model explains apparent enzyme activity loss at high temperatures that cannot be accounted for by irreversible thermal denaturation. The Equilibrium Model describes enzyme behavior in the presence of substrates and under assay conditions; thus its associated parameters, deltaHeq and Teq, may have physiological significance. The Equilibrium Model parameters have been determined for twenty-one enzymes of diverse origins. The results demonstrated the wide applicability of the Equilibrium Model to enzymes of different types and temperature affinity. The study has also established deltaHeq as the first quantitative measure of enzyme eurythermalism and demonstrated the relationship between Teq and optimal growth temperature of organisms. The Equilibrium Model is therefore a useful tool for studying enzyme temperature adaptation and its role in adaptations to thermophily and eurythermalism. Moreover, it potentially enables a description of the originating environment from the properties of the enzymes. The Equilibrium Model has been employed to characterize enzymes isolated from bacterial episymbionts of Alvinella pompejana. A. pompejana inhabits one of the most extreme environments known to science and has been proposed as an extremely eurythermal organism. A metagenomic study of the A. pompejana episymbionts has unveiled new information related to the adaptive and metabolic properties of the bacterial consortium; the availability of metagenomic sequences has also enabled targeted retrieval and heterologous expression of A. pompejana episymbiont genes. By inspecting enzymes derived from the unique episymbiotic microbial consortium intimately associated with A. pompejana, the study has shed light on temperature adaptations in this unique symbiotic relationship. The findings suggested that eurythermal enzymes are one of the mechanisms used by the microbial consortium to achieve its adaptations. By combining metagenomic and enzymological studies, the research described in this thesis has lead to insights on the eurythermalism of a complex microbial system from an enzymological aspect. The findings have enhanced our knowledge on how life adapts to extreme environments, and the validation of the Equilibrium Model as a tool for studying enzyme temperature adaptation paves the way for future studies.
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Stephenson, Anthony Aaron. "Mechanistic studies of enzymes involved in DNA transactions." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1531497128385619.
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Leksawasdi, Noppol Biotechnology & Biomolecular Sciences (BABS) UNSW. "Kinetics and modelling of enzymatic process for R-phenylacetylcarbinol (PAC) production." Awarded by:University of New South Wales. Biotechnology and Biomolecular Sciences (BABS), 2004. http://handle.unsw.edu.au/1959.4/20846.
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R-phenylacetylcarbinol (PAC) is used as a precursor for production of ephedrine and pseudoephedrine, which are anti-asthmatics and nasal decongestants. PAC is produced from benzaldehyde and pyruvate mediated by pyruvate decarboxylase (PDC). A strain of Rhizopus javanicus was evaluated for its production of PDC. The morphology of R. javanicus was influenced by the degree of aeration/agitation. A relatively high specific PDC activity (328 U decarboxylase g-1 mycelium) was achieved when aeration/agitation were reduced significantly in the latter stages of cultivation. The stability of partially purified PDC and crude extract from R. javanicus were evaluated by examining the enzyme deactivation kinetic in various conditions. R. javanicus PDC was less stable than Candida utilis PDC currently used in our group. A kinetic model for the deactivation of partially purified PDC extracted from C. utilis by benzaldehyde (0?00 mM) in 2.5 M MOPS buffer has been developed. An initial lag period prior to deactivation was found to occur, with first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration. A mathematical model for the enzymatic biotransformation of PAC and its associated by-products has been developed using a schematic method devised by King and Altman (1956) for deriving the rate equations. The rate equations for substrates, product and by-products have been derived from the patterns for yeast PDC and combined with a deactivation model for PDC from C. utilis. Initial rate and biotransformation studies were applied to refine and validate a mathematical model for PAC production. The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U carboligase ml-1. Michaelis-Menten kinetics were determined for the effect of pyruvate concentration on the reaction rate. The effect of benzaldehyde on the rate of PAC production followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde and acetoin. Implementation of digital pH control for PAC production in a well-stirred organic-aqueous two-phase biotransformation system with 20 mM MOPS and 2.5 M dipropylene glycol (DPG) in aqueous phase resulted in similar level of PAC production [1.01 M (151 g l-1) in an organic phase and 115 mM (17.2 g l-1) in an aqueous phase after 47 h] to the system with a more expensive 2.5 M MOPS buffer.
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Meriläinen, G. (Gitte). "Structural and enzymological studies of the thiolase enzymes." Doctoral thesis, University of Oulu, 2009. http://urn.fi/urn:isbn:9789514291982.
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Abstract
In the cells, the last step of the beta-oxidation cycle, aiming at the degradation of fatty acids, is catalyzed by the enzyme named thiolase. It shortens the acyl chain of the acyl-CoA by two carbons. The reaction is reversible, it can proceed for both directions. Thiolases are divided into two categories, synthetic and degradative ones. These two classes of thiolases differ not only by their biological function, but also by their substrate specificity. Degradative thiolases accept substrates with various lengths but synthetic thiolases only accept short chain-acyl-CoAs as a substrate.
In humans, at least six isozymes of thiolases are found. The mitochondrial biosynthetic thiolase, T2, differs from other thiolases by getting activated by potassium. In addition, it accepts branched acyl-CoA, namely 2-methyl-acetoacetyl-CoA, as a substrate. This molecule is an important reaction intermediate in the degradation of the amino acid isoleucine. Many human patients have been diagnosed to have a mutation in the gene of T2, and they are treated with a special diet.
The results of this theses show that potassium ion rigidifies the groups of the T2 protein involved in the substrate binding. The presence of potassium increases the reaction rate and it also raises the affinity towards some of the substrates.
The enzyme mechanistic studies with bacterial thiolase revealed that the oxyanion hole 1, formed by a water molecule and histidine side chain, is important for the synthetic reaction, not so much for the degradative direction. Binding studies showed that both the terminal sulfur of the substrate and the sulfur of the catalytic cysteine are important for the right positioning of the substrate. The electrostatics of the active site also have a significant role in the catalysis. These studies give a good basis for future studies aiming at drug development against this enzyme in pathogenic species.
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Mok, Yiu Ki. "The role of adsorbed enzymes in determining the hydrolysis kinetics of pretreated lignocellulosic biomass." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/52996.
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The cost-effective production of sugars from biomass continues to remain challenging, partly due to the relatively high enzyme/protein loading required to effectively hydrolyze pretreated lignocellulosic substrates. Previous works have shown conflicting observations regarding the correlation between enzyme adsorption and the hydrolytic performance of an enzyme mixture. Unfortunately, it has proven difficult to accurately determine the roles of adsorbed enzymes during the hydrolysis of lignocellulosic substrates, in part because of the interference that protein determination methods encounter from the release of sugars and other biomass derived materials, the lack of a hydrolysis strategy for hydrolysis with only adsorbed enzymes and the use of “model” substrates in many studies.
To better understand the role that adsorbed enzymes play in cellulose deconstruction, it is important that we are able to accurately quantify protein distribution and enzyme performance. Various protein quantification assays were initially assessed for their ability to accurately and reproducibly quantify protein/enzymes during typical biomass hydrolysis conditions. However, the ninhydrin assay, which was the most promising assay due to its specificity for protein and compatibility with most compounds derived from lignocellulosic samples, still suffered from the incompatibility with sugar degradation products, long hydrolysis times and potentially wide-ranging standard deviations. To overcome these limitations, an accurate and rapid modified ninhydrin assay was developed which employed a sodium borohydride treatment to eliminate sugar interference followed by acid hydrolysis at 130ºC, reducing the overall reaction time to 4 hours.
Utilizing the modified ninhydrin assay, the role of adsorbed enzymes in determining the rate and extent of hydrolysis of several different pretreated biomass substrates was then assessed. Once the distribution of enzymes reached equilibrium, after 60 minutes, those enzymes that were adsorbed or free in solution were separated by centrifugation and subsequently assessed for their ability to hydrolyze various cellulosic substrates at different enzyme loadings. It was apparent that the adsorbed enzymes were critically important as the removal of those enzymes in solution resulted in no significant decrease in the rate and extent of hydrolysis. By using the adsorbed enzymes, enzyme loadings could be reduced by up to 53% while resulting in similar hydrolysis yields.Forestry, Faculty of
Graduate
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Sharma, Narayan Prasad. "STRUCTURE/FUNCTION STUDIES ON METALLO-B- LACTAMASE ImiS FROM Aeromonas bv. sobria." Oxford, Ohio : Miami University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1181583976.
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Stockert, Amy L. "Spectroscopic and kinetic studies of bovine xanthine oxidase and Rhodobacter capsulatus xanthine dehydrogenase." Connect to this title online, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1089910515.
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Thesis (Ph. D.)--Ohio State University, 2004.Title from first page of PDF file. Document formatted into pages; contains xv, 172 p.; also includes graphics. Includes bibliographical references (p. 165-172).
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Evans, Geraint Wyn. "Real-time single-molecule observations of conformational changes in DNA polymerase." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:fdf11b59-2e58-4174-9219-9d61e4528f65.
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Genetic information is encoded in the long sequence of bases which form DNA, which is replicated during cell division by enzymes known as DNA Polymerases. Polymerases replicate DNA extremely accurately to avoid errors which can cause cell death and diseases such as cancer, although the mechanisms behind these extraordinary fidelities are not well understood. A large conformational change in the protein, in which the “fingers" subdomain closes around an incoming nucleotide, is thought to be implicated in these fidelity mechanisms. Here we present an assay to monitor this conformational change in single polymerase molecules, in real-time. We achieve this using total-internal-reflection-fluorescence (TIRF) microscopy to monitor the fluorescence resonance energy transfer (FRET) of an intra-protein dye labelled DNA Polymerase I (KF) as it binds to surface-immobilised DNA. Initially, we investigated the polymerase fingers-conformations during the pre-chemistry polymerisation reaction, resolving forward and backward rates which would be challenging to observe using ensemble techniques. These observations confirmed that KF closes rapidly around complementary nucleotide, but we discovered that the reverse step, fingers-opening, is particularly slow relative to chemistry. These finger kinetics act to remove the influence of the reaction rate-limiting step on fidelity, surprising given decades of investigations have focused on the rate-limiting step as the key determinant of fidelity. We also use our kinetic measurements to quantify contributions of different reaction steps to the macroscopic error rate of the polymerase. Subsequently, we developed our assay to investigate the fingers-conformations across the entire DNA polymerisation reaction. We observed single-nucleotide incorporations, and processive DNA polymerisation at high and low nucleotide concentrations, which suggested heterogeneous nucleotide incorporation rates. The observations demonstrated that the post-chemistry slow step that limits processive polymerisation occurs before post-chemistry fingers-opening, or is accounted for by post-chemistry fingers-opening. We observe a correlation in turn-over kinetics and binary complex kinetics, suggesting that turn-over rates could be limited by the intrinsic dynamics of the binary complex, as seen in other protein systems, although more work is needed on this.
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Andersson, C. Evalena. "Structure-Function Studies of Enzymes from Ribose Metabolism." Doctoral thesis, Uppsala University, Department of Cell and Molecular Biology, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3999.
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In the pentose phosphate pathway, carbohydrates such as glucose and ribose are degraded with production of reductive power and energy. Another important function is to produce essential pentoses, such as ribose 5-phosphate, which later can be used in biosynthesis of nucleic acids and cofactors.
This thesis presents structural and functional studies on three enzymes involved in ribose metabolism in Escherichia coli.
Ribokinase is an enzyme that phosphorylates ribose in the presence of ATP and magnesium, as the first step of exogenous ribose metabolism. Two important aspects of ribokinase function, not previously known, have been elucidated. Ribokinase was shown to be activated by monovalent cations, specifically potassium. Structural analysis of the monovalent ion binding site indicates that the ion has a structural rather than catalytic role; a mode of activation involving a conformational change has been suggested. Product inhibition studies suggest that ATP is the first substrate to bind the enzyme. Independent Kd measurements with the ATP analogue AMP-PCP support this. The results presented here will have implications for several enzymes in the protein family to which ribokinase belongs, in particular the medically interesting enzyme adenosine kinase.
Ribose 5-phosphate isomerases convert ribose 5-phosphate into ribulose 5-phosphate or vice versa. Structural studies on the two genetically distinct isomerases in E. coli have shown them to be fundamentally different in many aspects, including active site architecture. However, a kinetic study has demonstrated both enzymes to be efficient in terms of catalysis. Sequence searches of completed genomes show ribose 5-phosphate isomerase B to be the sole isomerase in many bacteria, although ribose 5-phosphate isomerase A is a nearly universal enzyme. All genomes contain at least one of the two enzymes. These results confirm that both enzymes must be independently capable of supporting ribose metabolism, a fact that had not previously been established.
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LEE, KANG-MIN. "Enzymes en milieux heterogenes." Université Louis Pasteur (Strasbourg) (1971-2008), 1989. http://www.theses.fr/1989STR13051.
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L'emploi des enzymes en synthese organique pose quelque probleme du fait de la faible solubilite des produits organiques dans l'eau. C'est pourquoi l'alcool dehydrogenase a ete cristallisee; les microemulsions offrent des proprietes favorables a l'activite enzymatique
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Tarhonskaya, Hanna. "Kinetic and mechanistic studies of oxygen sensing Fe(II)/2-oxoglutarate dependent oxygenases." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:94924981-8b73-43ba-9d90-47a73b84e5f9.
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The Fe(II)/2-oxoglutarate (2OG) dependent oxygenases are a widespread enzyme family, which are characterised by structurally similar active sites and proposed to employ a common reaction mechanism. The work described in this thesis concerned kinetic and biophysical studies on 2OG oxygenases, with a particular focus on the hypoxia-inducible transcription factor (HIF) hydroxylases and mechanistic aspects of their reaction with oxygen. The four human HIF hydroxylases regulate cellular levels and transcriptional activity of HIF by catalysing its post-translational hydroxylation in response to changes in oxygen availability. The three prolyl hydroxylase domain enzymes (PHDs1-3) and factor inhibiting HIF (FIH) are proposed to act as cellular oxygen sensors and provide a direct link between oxygen availability and the hypoxic response. Previous transient kinetic studies have shown that PHD2 (the most important human PHD isoform) reacts slowly with oxygen, a factor proposed to be related to its oxygen-sensing role. The molecular mechanisms for the slow PHD2 reaction with oxygen were investigated using a range of kinetic and biophysical techniques to probe the effects of key active site substitutions. The studies reveal that a conservative substitution to an Fe(II)/H2O binding residue results in 5-fold faster reaction with oxygen, suggesting a role for H2O release from the active site in limiting the ability of oxygen to react with PHD2. This thesis also describes the first transient kinetic studies of FIH. The obtained results show that the rate of the FIH reaction with oxygen was significantly faster than for PHD2. Further, FIH catalyses hydroxylation not only of HIF-α, but also of proteins containing ankyrin repeat domains (ARD). The rate of the FIH reaction with oxygen was shown to be substrate dependent; faster oxygen activation of the reaction in the presence of ARD compared with HIF substrates was observed. Mechanistic studies were performed to investigate a report that PHD2 is involved in the enzymatic oxidation of an oncometabolite (R)-2-hydroxyglutarate (2HG) to give 2OG, in what would be an unprecedented reaction for a 2OG oxygenase. This work found that 2HG does not substitute for 2OG in PHD2 catalysis. Instead, the non-enzymatic transformation of 2HG to 2OG was observed, which could potentially contribute to the reported 2HG-dependent PHD activation in vivo. The biophysical and transient kinetic techniques used for studying the HIF hydroxylases were also applied to study the mechanism of deacetoxycephalosporin C synthase (DAOCS, the enzyme catalysing penicillin N ring expansion). Previously, it has been suggested that the DAOCS mechanism differs from the consensus 2OG oxygenase mechanism. The results described in this thesis provide strong evidence that DAOCS employs the consensus ordered mechanism characteristic of 2OG oxygenases, supporting the proposal that the consensus mechanism is a common feature of the 2OG oxygenase family. Overall, the work described in this thesis is supportive of the proposal that most, if not all, 2OG oxygenases employ a common mechanism. However, the differences in the kinetics of their reaction with oxygen, presented throughout the thesis, suggest that different 2OG oxygenases have different rate-limiting steps. Thus, the kinetics of specific oxygenases may be adapted to their biological function, in particular that of PHD2 as the key cellular O2 sensor.
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Wirth, Petra. "Enzymes en solvants organiques." Grenoble 2 : ANRT, 1988. http://catalogue.bnf.fr/ark:/12148/cb37619244x.
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Costa, Rodrigo de Andrade. "Degradação enzimática de clorofenol em microrreator." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/3/3137/tde-29062016-161332/.
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O microrreator faz parte de conjunto de dispositivos de uma nova e promissora tecnologia, que podem ser chamados de micro fabricados, atuante em campos como a da química, biológica, farmacêutica, engenharia química e biotecnologia. Trata-se de um dispositivo que possibilita reação química, tais como os reatores convencionais, mas com dimensões menores, com canais na escala micrométrica. A tecnologia de miniaturização de dispositivos para reações químicas vem se expandindo promovendo uma importante evolução, com microssistemas que abrange dispositivos mais eficazes, com configuração e geometrias específicas e menor consumo de energia, onde reações com elevadas taxas de transporte podem ser usadas para muitas finalidades diferentes, tais como, reações rápidas, mistura, reações sensíveis à temperatura, temperatura de homogeneização, ou até mesmo precipitação de nano partículas. Devido sua escala ser extremamente reduzida em relação à escala macro, oferecem um sistema que permite uma investigação do processo em um curto espaço de tempo, sendo muito útil para o rastreio de substratos, enzimas, condições de reação, bem como a determinação de parâmetros cinéticos. O presente trabalho teve por objetivo estudar a biodegradação enzimática de 2,4,6-Triclorofenol, com a utilização das enzimas Lacase e Soybean Peroxidase em microrreator da Syrris com volume de 250 ?l, que permite o estudo de cinéticas muito rápidas. Para as análises de degradação utilizou-se duas enzimas, a Lacase em concentrações de 0,05; 0,1 e 0,2 mg/ml; e a Soybean Peroxidase em concentrações de 0,0005; 0,001 e 0,002 mg/ml com a adição de Peróxido de Hidrogênio. Através dos ensaios realizados obteve-se dados experimentais da reação enzimática, possibilitando a verificação da taxa inicial de reação e sua cinética. Posteriormente, realizou-se as análises em simulação utilizando os dados experimentais, que através de um sistema de EDOs estimando inicialmente as constantes cinéticas k1, k2 e k3 usando a ferramenta ESTIMA, onde apresentaram duas respostas, uma resposta típica de mínimos quadrados, e a outra resposta que a velocidade inicial, que foi melhor representada pelos parâmetros obtidos. O método empregado na degradação do substrato, o microrreator mostrou-se eficiente, permitindo a detecção de baixo consumo de substrato para a determinação da taxa inicial, em curto tempo de residência. Perante os ensaios realizados com Lacase e Soybean Peroxidase, o microrreator é também um equipamento eficaz na repetitividade e na reprodutibilidade dos dados obtidos em diferentes concentrações.The microreactor is part of a set of devices in a new and promising technology, which can be called micro manufactured, active in fields such as chemical, biological, pharmaceutical, chemical engineering and biotechnology. It is a device that enables chemical reactions, such as conventional reactors, but with smaller dimensions, in the micrometer scale channels. Miniaturization technology devices for chemical reactions is expanding promoting an important development, with microsystems covering most effective devices, configuration and specific geometries and lower power consumption, where reactions with high transportation fees can be used for many different purposes such as fast reactions, mixing, temperature sensitive reactions, homogenization temperature or even precipitation of nanoparticles. Because of its scale is greatly reduced compared to the macro scale, provide a system which allows an investigation of the process in a short time, being very useful for screening for substrates, enzymes, reaction conditions, and the determination of kinetic parameters. One of the advantages of using microreactors is that this equipment requires small amounts of reagents for performing a catalytic reaction of action, and is very important when dealing with enzyme as a catalyst. This study aimed to study the enzymatic biodegradation of 2,4,6-Trichlorophenol with the use of laccase and Soybean Peroxidase enzymes in microreactor Syrris with volume of 250 ?l, which allows the study of very fast kinetics. For degradation analyzes were used two enzymes, laccase concentrations of 0.05; 0.1 and 0.2 mg / ml; and Soybean peroxidase at concentrations of 0.0005; 0.001 and 0.002 mg / ml with the addition of Hydrogen Peroxide. Through trials was obtained experimental data from enzyme reaction, allowing the verification of the initial reaction rate and its kinetics. Later, there was the analysis simulation using the experimental data, which through a system of ODEs initially estimating the rate constants k1, k2 and k3 using the ESTIMA tool, which had two answers, a typical response of least squares, and another answer to the initial rate, which was best represented by the parameters obtained. The method used in substrate degradation, the microreactor was efficient, allowing low substrate consumption detection for determining the initial rate in the short residence time. Before the tests with Laccase and Soybean Peroxidase, the microreactor is also an effective equipment in the repeatability and reproducibility of the data obtained at different concentrations.
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Kartal, Önder. "The role of interfacial and 'entropic' enzymes in transitory starch degradation : a mathematical modeling approach." Phd thesis, Universität Potsdam, 2011. http://opus.kobv.de/ubp/volltexte/2011/5394/.
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Plants and some unicellular algae store carbon in the form of transitory starch on a diurnal basis. The turnover of this glucose polymer is tightly regulated and timely synthesis as well as mobilization is essential to provide energy for heterotrophic growth. Especially for starch degradation, novel enzymes and mechanisms have been proposed recently. However, the catalytic properties of these enzymes and their coordination with metabolic regulation are still to be discovered.
This thesis develops theoretical methods in order to interpret and analyze enzymes and their role in starch degradation. In the first part, a novel description of interfacial enzyme catalysis is proposed. Since the initial steps of starch degradation involve reactions at the starch-stroma interface it is necessary to have a framework which allows the derivation of interfacial enzyme rate laws. A cornerstone of the method is the introduction of the available area function - a concept from surface physics - to describe the adsorption step in the catalytic cycle. The method is applied to derive rate laws for two hydrolases, the Beta-amylase (BAM3) and the Isoamylase (DBE/ISA3), as well as to the Glucan, water dikinase (GWD) and a Phosphoglucan phosphatase (DSP/SEX4).
The second part uses the interfacial rate laws to formulate a kinetic model of starch degradation. It aims at reproducing the stimulatory effect of reversible phosphorylation by GWD and DSP on the breakdown of the granule. The model can describe the dynamics of interfacial properties during degradation and suggests that interfacial amylopectin side-chains undergo spontaneous helix-coil transitions. Reversible phosphorylation has a synergistic effect on glucan release especially in the early phase dropping off during degradation. Based on the model, the hypothesis is formulated that interfacial phosphorylation is important for the rapid switch from starch synthesis to starch degradation.
The third part takes a broader perspective on carbohydrate-active enzymes (CAZymes) but is motivated by the organization of the downstream pathway of starch breakdown. This comprises Alpha-1,4-glucanotransferases (DPE1 and DPE2) and Alpha-glucan-phosphorylases (Pho or PHS) both in the stroma and in the cytosol. CAZymes accept many different substrates and catalyze numerous reactions and therefore cannot be characterized in classical enzymological terms. A concise characterization is provided by conceptually linking statistical thermodynamics and polymer biochemistry. Each reactant is interpreted as an energy level, transitions between which are constrained by the enzymatic mechanisms. Combinations of in vitro assays of polymer-active CAZymes essential for carbon metabolism in plants confirmed the dominance of entropic gradients. The principle of entropy maximization provides a generalization of the equilibrium constant. Stochastic simulations confirm the results and suggest that randomization of metabolites in the cytosolic pool of soluble heteroglycans (SHG) may contribute to a robust integration of fluctuating carbon fluxes coming from chloroplasts.Stärke hat eine herausragende Bedeutung für die menschliche Ernährung. Sie ist ein komplexes, wasserunlösliches Glucosepolymer und dient - als eine der wichtigsten Speicherformen von Kohlenhydraten in Pflanzen - der Aufrechterhaltung des Energiestoffwechsels. Unterschiedliche Organe enthalten Stärke. In Knollen und Samen wird die sogenannte Speicherstärke über lange Zeiträume auf- und abgebaut. Die im Allgemeinen weniger bekannte transitorische Stärke in Blättern und einigen einzelligen Algen wird in einem täglichen Rhythmus umgesetzt: Sie wird während der Photosynthese aufgebaut und in der Nacht abgebaut. Experimentelle Studien haben nachgewiesen, dass die Fähigkeit der Pflanze, den Abbau transitorischer Stärke zu regeln, essentiell ist, um während der Nacht das Wachstum der Pflanze zu gewährleisten. Da die Geschwindigkeit von biochemischen Reaktionen über Enzyme reguliert wird, ist die Aufklärung ihrer Funktion im Stoffwechsel eine notwendige Voraussetzung, um den komplexen Prozess des Wachstums zu erklären. Die vorliegende Arbeit stellt einen Versuch dar, die Funktion von Enzymen beim Stärkeabbau anhand von mathematischen Modellen und Computersimulationen besser zu verstehen. Dieser Ansatz erlaubt es, Eigenschaften des Systems durch Abstraktion anhand eines idealisierten Abbildes herzuleiten. Die mathematisch notwendigen Folgerungen dienen der Aufstellung von Hypothesen, die wiederum mit experimentellen Resultaten konfrontiert werden können. Stoffwechselsysteme sind komplexe Untersuchungsobjekte, bei denen eine rein qualitative Argumentation schnell an Grenzen gerät, wo mathematische Methoden die Möglichkeit von Aussagen noch zulassen. Der erste Teil der Arbeit entwickelt einen theoretischen Rahmen, um Gleichungen für die Geschwindigkeit oberflächenaktiver Enzyme herzuleiten. Dies ist notwendig, da die ersten Reaktionen, die dem Stärkeabbau zugeordnet werden, an ihrer Oberfläche stattfinden. Die Methode wird auf vier essentielle Enzyme angewandt: zwei abbauende Enzyme (Beta-Amylase und Isoamylase) und zwei den Abbau unterstützende Enzyme (Alpha-Glucan,Wasser-Dikinase und Phosphoglucan Phosphatase). Der zweite Teil entwickelt ein kinetisches Modell des Stärkeabbaus unter Verwendung der hergeleiteten Ratengleichungen. Das Modell bildet die Dynamik des Systems realistisch ab und legt nahe, dass ein spontaner Phasenübergang an der Oberfläche von geordneten zu weniger geordneten Zuständen stattfindet. Ferner wird die Hypothese aufgestellt, dass die reversible Modifikation der Oberfläche durch Enzyme besonders in der Anfangsphase des Abbaus einen synergetischen Effekt hat, d.h. den Abbau enorm beschleunigt. Dies könnte beim schnellen Umschalten von Stärkeaufbau zu Stärkeabbau regulatorisch relevant sein. Im letzten Teil werden kohlenhydrataktive Enzyme betrachtet, die in der löslichen Phase die Produkte des Stärkeabbaus weiterverarbeiten. Da diese sogenannten Transferasen auch in vielen anderen Organismen und Stoffwechselwegen vorkommen, wird ein allgemeiner Standpunkt eingenommen. Anhand von Methoden aus der statistischen Physik wird theoretisch wie experimentell nachgewiesen, dass diese Enzyme spontan die Entropie innerhalb des Stoffwechselsystems erhöhen. Diese Neigung, "Unordnung" zu schaffen, wird vom Organismus aber paradoxerweise ausgenutzt, um die Weiterverarbeitung von Kohlenhydraten im Stärkestoffwechsel zu stabilisieren. Dieser Mechanismus eröffnet einen neuen Blick auf energie- und entropiegetriebene Prozesse in Zellen.
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Nutt, Anu. "Hydrolytic and Oxidative Mechanisms Involved in Cellulose Degradation." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6888.
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Kaplan, Elise. "Aminoglycoside modifying enzymes involved in antibiotic resistance : functional and structural studies." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTT006.
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L'émergence de bactéries résistantes aux antibiotiques constitue un problème majeur de santé publique responsable d'un nombre croissant de décès, surtout dans les hôpitaux. La résistance aux aminoglycosides est principalement due à l'expression d'enzymes capables de les modifier, comme les aminoglycosides phosphotransférases (APH).Le premier volet de ce travail de thèse vise à mieux comprendre les bases moléculaires des interactions protéine-ligands et de la catalyse enzymatique d'une de ces enzymes, l'APH(2”)-IVa. La spécificité de substrats a été caractérisée en détails pour différents aminoglycosides par des méthodes thermodynamiques, de mesures cinétiques à l'état stationnaire et transitoire, par amarrage moléculaire et cristallographie aux rayons X. La seconde partie de cette étude consiste à développer et optimiser des inhibiteurs allostériques de ces enzymes capables de restaurer l'efficacité des aminoglycosides. Pour cela, une cavité, potentiellement impliquée dans la dynamique de l'APH(2”)-IVa, a été identifiée à partir de simulations de dynamique moléculaire. Celle-ci a servi de cible pour cribler, in silico, 12 000 composés issus de la banque de données Zinc. Ainsi, 14 composés ont été testés in vitro pour leur capacité à diminuer l'activité enzymatique d'APH. Parmi ces derniers, une molécule s'est révélée être un inhibiteur non-compétitif de l'APH(2”)-IVa. Une étude des relations structure-fonction a permis de déterminer les groupements les plus favorables à l'inhibition et d'identifier un composé plus efficace. L'utilisation de ces deux molécules permet de restaurer, par exemple, la sensibilité à la sisomicine d'une souche d'E. faecium exprimant cette enzyme. Cette étude fournit des bases au développement de thérapies combinant un aminoglycoside et un inhibiteur des enzymes d'inactivation constituant une stratégie pour lutter contre la résistance aux antibiotiques dans un contexte thérapeutiqueEmergence of multi-drug resistant bacteria leads to increasing fatal issues especially in hospitals. Resistance to aminoglycoside antibiotics is mainly due to the expression of modifying enzymes, such as aminoglycoside phosphotransferases (APH). The first aim of this project was to elucidate the molecular basis of protein-ligand interactions and catalysis of one of these enzymes, the APH(2”)-IVa. Promiscuity of aminoglycoside substrates has thus been characterized in details using thermodynamics, transient and steady state kinetics, molecular docking and X-ray crystallography techniques.The second part aimed to develop and optimize allosteric inhibitors of these enzymes able to counterbalance aminoglycoside resistance. For this purpose, a small cavity, potentially involved in APH dynamics, was identified from molecular dynamic simulations. This cavity was used as a target to virtually screen 12 000 compounds of the Zinc database. The efficiency of the 14 high-ranked molecules to inhibit APH was evaluated in vitro and lead to the identification of a non-competitive inhibitor of APH(2”)-IVa. Structure-activity relationships highlighted the most favourable substituents for APH inhibition and permitted to obtain a more potent compound. The two molecules were able to restore, for example, sisomicin susceptibility of an E. faecium strain, expressing this enzyme.This study provides a basis for the development of combined chemotherapies (antibiotic with enzyme inhibitor) which may overcome antibiotic resistance in a clinical context
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Menon, Navya. "Kinetics and structure-guided characterisation and engineering of aldehyde deformylating oxygenase (ADO) for a renewable microbial biofuel platform." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/kinetics-and-structureguided-characterisation-and-engineering-of-aldehyde-deformylating-oxygenase-ado-for-a-renewable-microbial-biofuel-platform(a1475b84-ae69-4488-bcc4-c840d662af4a).html.
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The increased demand for an alternative form of fuel has raised a great interest towards exploring various metabolic pathways and enzymes in several microbial species for hydrocarbon production. In recent years, cyanobacteria have emerged as an attractive microbial host and cyanobacterial metabolic pathways were targeted for engineering to produce "drop in" fuels such as propane and butane. Whilst appealing, practicalities for producing biofuels in cyanobacteria remain challenging, requiring the identification and engineering of natural biocatalysts and their integration into metabolic processes. Cyanobacterial hydrocarbon biosynthesis arises from fatty acid metabolism involving a potential enzyme, aldehyde deformylating oxygenase (ADO), which catalyses the decarbonylation of long-chain fatty aldehydes to alkanes, mainly in the conversion of octadecanal (C17H35CHO) to heptadecane (C17H36) and formate. The substrate specificity and preferences for long-chain aldehyde by ADO necessitates a detailed kinetic and structural characterisation in order to optimise/engineer this enzyme for future biotechnological applications. Thus, the main objective was to identify a potential ADO enzyme that can be optimised for shorter chain alkane production. By studying the substrate specificity and reaction kinetics of different ADO enzymes, it was found that ADO from Prochlorococcus marinus MIT 9313 (PmADO) is a potential target for short chain alkane production. The crystal structural of PmADO was solved and further GC-MS analysis was carried out to identify the chemical origin of a mixture of long-chain fatty acid in the active site, originated from E. coli cells during recombinant over-expression and purification. It was suggested that the structure-guided protein engineering for short-chain alkane production should be carried out along with the removal of this adventitious ligand from the active site in order to increase the alkane production. Four important residues present at the entrance of the ligand-binding cavity were targeted and saturated mutagenesis was performed on PmADO to identify variants that excluded the long fatty acid ligands from the active site but have specificity and higher conversion rates for shorter chain aldehydes. This identified two variants, V41Y and A134F, with the A134F variant that not only exhibiting an improved activity and turnover value of PmADO by four-fold but also improved binding affinity for butyraldehyde by 2 times. Finally the improved variants were incorporated in a host organism (E. coli) and the possibilities for the development of a microbial platform for renewable propane synthesis based on a fermentative clostridial butanol pathway were explored. Four pathways were designed namely atoB-adhE2, atoB-TPC7, nphT7-adhE2 and nphT7-TPC7 routes, which utilise CoA intermediates selected to incorporate ADO as the terminal enzyme. When PmADO was co-expressed with these pathways, the engineered E. coli host produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed A134F variant ofPmADO with an enhanced specificity towards short-chain substrates, and (iii) including a ferredoxin-based electron supply system, the propane titre was increased up to 3.40 ± 0.19 mg/L. It was also shown that the best propane producing pathways are scalable in a 250 mL flask and in a large-scale (up to 30 L) fermentor setup. This thesis focuses on the detailed kinetics and structure-guided characterisation and engineering studies on the ADO enzyme for the development of a renewable microbial biofuel platform.
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Geitmann, Matthis. "Biosensor Studies of Ligand Interactions with Structurally Flexible Enzymes : Applications for Antiviral Drug Development." Doctoral thesis, Uppsala universitet, Institutionen för naturvetenskaplig biokemi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5797.
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The use of a surface plasmon biosensor fills a missing link in kinetic studies of enzymes, since it measures directly the interaction between biomolecules and allows determination of parameters that are determined only indirectly in activity assays. The present thesis deals with kinetic and dynamic aspects of ligand binding to two viral enzymes: the human cytomegalovirus (HCMV) protease and the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). The improved description of interactions presented herein will contribute to the discovery and development of antiviral drugs. The biosensor method provided new insights into the interaction between serine proteases and a peptide substrate, as well as substrate-induced conformational changes of the enzymes. The direct binding assay served as a tool for characterising the binding mechanism of HCMV protease inhibitors. Kinetic details of the interaction between HIV-1 RT and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were unravelled. The recorded sensorgrams revealed several forms of complexity. A general binding model for the analysis was derived from the data, describing a two-state mechanism for the enzyme and a high- and a low-affinity interaction with the inhibitor. Interaction kinetic constants were determined for the clinically used NNRTIs and several investigational inhibitors. The established method was applied to investigate the mechanism of resistance against NNRTIs. Amino acid substitutions in the NNRTI-binding site resulted in both decreased association rates and increased dissociation rates for the inhibitors. The K103N and the L100I substitution also interfered with the formation of the binding site, thereby facilitating inhibitor binding and unbinding. Finally, thermodynamic analysis revealed that, despite the hydrophobic character of the interaction, NNRTI binding was mainly enthalpy-driven at equilibrium. Large entropy contributions in the association and dissociation indicated that binding is associated with a dynamic effect in the enzyme.
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Bauman, Mariia A. "Characterizing the Effect of Conformational Changes in the Protein SufU on its Ability to Enhance Enzymatic Activity of the Cysteine Desulfurase SufS in Streptococcus mutans." Bowling Green State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1467906490.
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Ma, Guihua. "The Kinetics, Biochemical Patterns, and Microbial Ecology in Multiredox (Anoxic, Microaerobic, Aerobic) Activated Sludge Systems Treating BTX Containing Wastewater." Diss., Virginia Tech, 1999. http://hdl.handle.net/10919/28883.
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BTX biodegradation rates, biochemical expression patterns and microbial ecology were studied under anoxic (denitrifying), anoxic/microaerobic/aerobic, and anoxic/microaerobic conditions in activated sludge sequencing batch reactors. The studies showed that toluene and m-xylene were denitrified via benzoyl-CoA reductase. Although benzene, o-, and p-xylene were recalcitrant under denitrifying conditions, they were biodegraded under microaerobic (< 0.2 mg/L dissolved oxygen) and nitrate or nitrite (NOx)-supplemented microaerobic conditions. The patterns of the specific enzymes associated with BTX biodegradation under microaerobic conditions indicated that the three compounds were metabolized by oxygen-dependent pathways. The expression levels of catechol 1, 2-dioxygenase and catechol 2, 3-dioxygenase under microaerobic conditions were induced to levels as high as under aerobic conditions (> 4 mg/L dissolved oxygen). Benzene, o-, and p-xylene biodegradation rates were twice as fast under NOx-supplemented compared to NOx-free microaerobic conditions, and the specific biodegradation rates under aerobic and NOx-supplemented microaerobic conditions were comparable.
16S rRNA probes targeting representative toluene-degraders were used to investigate the microbial communities in the three sequencing batch reactors by using a dot blot hybridization technique. The hybridization results suggest that multiple redox environments fostered a more diverse microbial community and the activities of the target organisms in the reactors with multiple redox environments were higher than in the single redox reactor. Additionally, facultative toluene-degraders appeared to play a less significant role than the strict anoxic and aerobic toluene-degraders in all three SBRs.Ph. D.
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Kaganjo, James Chege. "Structure-function studies of 5-aminolevulinic acid (ALA) synthases." Bowling Green State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1510583488812729.
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Johnson, Eachan Oliver Daniel. "Protein-protein recognition in biological systems exhibiting highly-conserved tertiary structure : cytochrome P450." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:d19f5f52-d1ce-4ec2-be83-fd52f01124f8.
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Protein tertiary structure is more conserved than amino acid sequence, leading to a diverse range of functions observed in the same fold. Despite < 20 % overall sequence identity, cytochromes P450 all have the same fold. Bacterial Class I P450s receive electrons from a highly specific, often unidentified, ferredoxin, in which case the hemoprotein is termed “orphaned”. CYP199A2, a Class I P450, accepts electrons from ferredoxins Pux and HaPux. Five orientation-dependent and one orientation-independent DEER measurements on paramagnetic HaPux and spin-labelled CYP199A2 yielded vector restraints, which were applied to building a model of the CYP199A2:HaPux complex in silico. A different binding mode was observed compared to P450cam:Pdx and P450scc:Adx, both recently elucidated by X-ray crystallography. This protocol was also applied to the CYP101D1:Arx complex. The first three measurements indicate that this heterodimer does not have a similar orientation to CYP199A2:HaPux, P450cam:Pdx, or P450scc:Adx. P450cam was fused to putidatredoxin reductase (PdR) to explore the kinetic effects with a view to improving electron transfer to orphan P450s. Heme incorporation of this enzyme depends on linker length. In whole cells, the fusion was more active after longer incubations. In vitro kinetics of the fusion exhibited some co-operativity and enhanced kinetics over the unfused system under steady-state conditions. The putative iron-sulfur biosynthesis ferredoxin PuxB had been engineered by rational mutagenesis to support catalysis by CYP199A2. It was confirmed this arose from improved protein-protein recognition. Engineering of E. coli ferredoxin based on these findings was carried out, resulting in electron-transfer to CYP199A4 from a novel engineered alien ferredoxin.
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DURAND-PAIRE, GENEVIEVE. "Enzymes hydrolytiques chez dictyostelium discoideum. Localisation dans la voie d'endocytose et mecanismes de secretion." Université Joseph Fourier (Grenoble), 1996. http://www.theses.fr/1996GRE10164.
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L'endocytose est une fonction que l'on retrouve chez la plupart des eucaryotes et qui assure l'entree dans la cellule des nutriments. L'amibe dictyostellium discoideum est un modele de choix pour l'etude de l'endocytose puisque ce processus y est actif et constitue son seul mode de nutrition. La degradation du materiel ingere a longtemps ete consideree comme un evenement lysosomal. Cependant, des resultats montrent la presence d'hydrolases dans des compartiments plus precoces. Les cinetiques d'entree realisees avec la serum albumine fluorescente montrent la presence de peptides 10 minutes apres l'internalisation les glycosidases (hexosaminidase, glucosidase et mannosidase) et la phosphatase acide ont ete localisees dans des compartiments situes a moins de 5 minutes du point d'entree. Le lysosome est atteint apres 15 minutes apres l'internalisation du fluide. Ceci implique donc la presence de proteases, de glycosidases et de phosphatase acide dans des compartiments endosomaux. Dictyostelium secrete constitutivement et selectivement une fraction de ses enzymes lysosomales dans le milieu externe, a l'exception des cathepsines. Des mutants resistants au methylene-diphosphonate, qui sont incapables de maintenir le gradient de ph entre leur voie endocytaire et le cytoplasme, ont un contenu en enzymes hydrolytiques reduit. Mimer le defaut d'acidification chez la souche parentale par ajout de bases faibles ou d'un inhibiteur de l'atpase vacuolaire (bafilomycine) ne reproduit pas le defaut enzmatique. La vacuolisation des cellules mutantes pourraient etre un facteur conduisant a la secretion
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Fernandez, Christine Cheryl. "Characterisation of cytochrome P450 azole drug-resistant sterol demethylase CYP51B1 and expression of CYP123 and CYP136 from Mycobacterium tuberculosis." Thesis, University of Manchester, 2011. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:171502.
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Tuberculosis (TB) affects nearly a third of the world’s population and has been termed a ‘Global Emergency’ by the WHO. The emergence of multi/extensively drug resistant (M/XDR) strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB, and the increasing incidences of azole drug resistant sterol demethylases (CYP51) from pathogenic fungi has propelled studies to understand mechanisms of azole drug resistance on the drug target CYP51. Since Mtb is devoid of a sterol biosynthetic pathway, the presence and study of CYP51B1 and 19 other Cytochrome P450s in its genome is important to clarify host-pathogen mechanism of infection and the potential of using azole drugs to treat TB. In this study, CYP51B1 from Mtb was used as the model enzyme to study CYP51 mutants from Candida albicans fluconazole-resistant clinical strains. By protein engineering methods, F89H, L100F, S348F, G388S and R391K CYP51B1 mutants were made and azole drug binding properties were investigated using stopped-flow kinetics and static equilibrium methods. Dissociation constant (Kd) values were derived for a range of commercially available azole drugs by fitting the equilibrium binding data to a hyperbolic equation. Kd values for stopped-flow kinetics were derived by plotting observed binding rates (kobs) across different azole drug concentrations against time, followed by fitting multiple kobs data to a linear equation to derive azole drug de-binding (koff) and binding (kon) rate constants – the Kd was obtained by koff/kon. Extinction coefficient for heme b content in mutants and Wild Type (WT) CYP51B1 were an average of ɛ419 = 96.1 mM-1 cm-1. Biochemical characterisation of the mutants were carried out using established experiments on CYP51 – reduction of Fe(III)-heme to Fe(II)-heme, NO binding to Fe(III)-heme, rates of CO-Fe(II) adduct formation and rates of collapse of the P450 to P420 species in the presence of CO and estriol with redox partners from Mtb. In order to elucidate the effects of the above mutations on the iron-heme catalytic region, electron paramagnetic resonance (EPR) experiments were carried out with and without azole drugs. Circular dichroism (CD), differential scanning calorimetry (DSC) and multi-angled laser light scattering (MALLS) analysis confirmed that F89H, R391K and L100F mutants were stable and homogeneous. Crystallogenesis was successful for the above mentioned mutants and atomic structures were obtained for all mutants and WT CYP51B1 (in ligand-bound and substrate-free forms), except for S348F and G388S mutants which were expressed as inclusion bodies and 60% holoenzyme, respectively. Reconstituted catalytic assays to determine the sterol demethylating propensity of the mutants were carried out using redox partners from Mtb or E. coli, and with lanosterol and dihydrolanosterol as the surrogate substrates. Redox potentiometry showed similar potentials to WT for all mutants except for the G388S mutant which was relatively positive (–102 mV). Redox cycling experiments followed by EPR analysis for mutants and WT resulted in a novel P450 high-spin species at g value 5.84 (80 %) which gradually collapsed to the initial low spin state over 48 h. Expression trials were concurrently carried out on two other Mtb P450 genes – CYP123 (Rv0744c) and CYP136 (Rv3059) products of which may have similar functions to CYP51B1 or may share similar redox partners. CYP123 is located on the same operon as CYP51B1 while CYP136 has a 29% sequence identity to another CYP51 from a marine slime bacterium. Although further work is necessary, in this study CYP123 was expressed totally as inclusion bodies while CYP136 was expressed as soluble apoprotein fused with trigger factor chaperone.
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Sha, Zhou. "A Chemical Approach to Detect and Characterize The Activities of Mitochondrial ATP-dependent Protease Lon and ClpXP." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1595239191845497.
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Molefe, Duduzile Mabel. "Studies directed towards the synthesis of chromone carbaldehyde-derived HIV-1 protease inhibitors." Thesis, Rhodes University, 2008. http://hdl.handle.net/10962/d1015542.
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A series of chromone-3-carbaldehydes have been prepared using Vilsmeier-Haack methodology while a corresponding series of chromone-2-carbaldeydes have been synthesized via the Kostanecki-Robinson reaction. Baylis-Hillman reactions have been conducted on both series of chromone carbaldehydes using three different catalysts, viz., 1,4-diazabicyclo(2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec- 7-ene (DBU) and 3-hydroxyquinuclidine (3HQ), and acrylonitrile, methyl acrylate and methyl vinyl ketone as the activated alkenes. These reactions have typically (but not always!) afforded both normal Baylis-Hillman and dimeric products. Attention has also been given to the use of 1-methyl-2-pyrrolidine (1-NMP), an ionic liquid, to replace normal organic solvents, and it has been found that, in the presence of DABCO, chromone-3-carbaldehydes afford the dimeric products alone. Reactions of chromone-3-carbaldehydes with methyl vinyl ketone have yielded unexpected, novel adducts, which appear to arise from preferential attack at C(2) in the chromone nucleus. Research on chromone-2-carbaldeydes under Baylis-Hillman conditions has also resulted in the formation of some interesting products instead of the expected Baylis-Hillman adducts. The Baylis-Hillman products have been explored as substrates for aza-Michael reactions using various amino derivatives including protected amino acids in the presence of the tetrabutylammonium bromide (TBAB) and the ionic liquid, 3-butyl-1- methylimidazoleboranetetrafluoride (BmimBF₄), as catalysts. The aza-Michael products have been targeted as truncated ritonavir analogues for investigation as potential HIV -1 protease inhibitors, and representative compounds have been subjected to enzyme inhibition assays to explore the extent and type of inhibition. Lineweaver-Burk and Dixon plots have indicated competitive inhibition in one case as well as non-competitive inhibition in another, and the inhibition constants (Ki) have been compared with that of the ritonavir. Computer modelling studies have also been conducted on selected chromonecontaining derivatives, using the ACCELRYS Cerius² platform. Interactive docking of the chromone-containing ligands into the HIV -1 protease receptor site, using the Ligandfit module, has indicated the importance of hydrogen-bonding interactions mediated by bridging water molecules situated in the receptor cavity. NMR spectroscopy has been used to elucidate complex and competing mechanistic pathways involved in the Baylis-Hillman reactions of selected 2-nitrobenzaldehydes with MVK in the presence of DABCO - reactions which afford the normal BaylisHillman product, the MVK dimer and syn- and anti-Baylis-Hillman type diadducts. The kinetic data confirm the concomitant operation of two pathways and reveal that, in the initial stage of the reaction, the product distribution is kinetically controlled, whereas in the latter stage, thermodynamic control results in the consumption of the normal Baylis-Hillman product and predominance of the anti-diadduct.
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Zaman, Flora. "Kinetics of enzyme models." Thesis, University of Kent, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263701.
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Byarugaba-Bazirake, George William. "The effect of enzymatic processing on banana juice and wine." Thesis, Link to the online version, 2008. http://hdl.handle.net/10019/1633.
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Johnson, Adam L. "Characterization of a Novel Protease in Staphylococcus aureus." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3943.
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A newly discovered cysteine protease, Prp, has been shown to perform an essential, site-specific cleavage of ribosomal protein L27 in Staphylococcus aureus. In Firmicutes and related bacteria, ribosomal protein L27 is encoded with a conserved N-terminal extension that must be removed to expose residues critical for ribosome function. Uncleavable and pre-cleaved variants were unable to complement an L27 deletion in S. aureus, indicating that this N-terminal processing event is essential and likely plays an important regulatory role. The gene encoding the responsible protease (prp) has been shown to be essential, and is found in all organisms encoding the N-terminal extension of L27. Cleavage of L27 by Prp represents a new target for potential antibiotic therapy. In order to characterize this protease, Prp has been overexpressed and purified. Using an assay we have developed, based on cleavage of a fluorogenic peptide derived from the conserved L27 cleavage sequence, we have undertaken an analysis of the enzyme kinetics and substrate specificity for Prp cleavage and tested predictions made based on a structural model using active-site mutants.
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Kakkar, Tarundeep Singh. "Theoretical studies on enzyme inhibition kinetics." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/289017.
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Enzyme inhibition studies are conducted to characterize enzymes and to examine drug-drug interactions. To characterize the inhibitory process (competitive, non-competitive and uncompetitive) and to determine the inhibitory constant (Kᵢ), data analysis techniques (e.g., Dixon, Lineweaver-Burk, etc.) are used to linearize the inherently non-linear rate of substrate metabolism vs. substrate concentration data. These techniques were developed before the general use of computers. However, many investigators still rely on these techniques in spite of the easy availability of non-linear regression fitting programs. In Chapter 2, three methods (simultaneous nonlinear regression fit (SNLR); Dixon; non-simultaneous, nonlinear fit [K(m,app)]) were compared for estimating Ki from simulated data sets generated from a competitive inhibition model equation with 10% CV added random error to the data values. Of the three methods, the SNLR method was found to be the most robust, the fastest and easiest to implement. The K(m,app) method also gave good estimates but was more time-consuming. The Dixon method failed to give accurate and precise estimates of Kᵢ. The purpose of the study in Chapter 3 was to examine the minimal experimental design needed to obtain reliable and robust estimates of Kᵢ (as well as V(max) and K(m)). Four cases were examined. In the experimental design that relied upon the least amount of data, a control data set was fit simultaneously with one of the substrate-inhibitor pairs (25-10 or 250-100 μM). A total of 4 rate values were analyzed per fit (i.e., 3 control + 1 inhibitor value). A total of 100 data sets were fit per substrate-inhibitor pair. The preceding was repeated for a random error of 20 %CV. Thus, the total number of experiments was reduced from 108 (in Chapter 2) to 12 (in Chapter 3) (Case IV). Good estimates of the enzyme kinetic parameters were obtained. In Chapter 4, the ability of the SNLR method to identify the correct mechanism of inhibition was evaluated; competitive or noncompetitive enzyme-inhibition. Two experimental designs were examined ("conventional, non-optimal" and "semi-minimal"). The semi-minimal design was successful in discriminating between the two enzyme-inhibition mechanisms even for data with 30 %CV added random error.
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Bayram, Mustafa. "Computer algebra approaches to enzyme kinetics." Thesis, University of Bath, 1993. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357810.
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Guo, Qi. "Enzyme dynamics and their role in formate dehydrogenase." Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/2216.
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How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.
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Astier, Yann. "Enzyme kinetics and electrochemical polymer transistor detection of enzyme reactions." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273800.
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Tsoumpra, Maria. "Targeting the mevalonate pathway for pharmacological intervention." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:fe945074-e645-4c1d-9598-e28b51a43cca.
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Farnesyl pyrophosphate synthase (FPPS) is a key branch point enzyme in the mevalonate pathway and the main molecular target of nitrogen-containing bisphosphonates (N-BPs), potent inhibitors of osteoclastic activity and the leading drug of choice for conditions characterized by excessive bone resorption. The main aim of this thesis is to investigate the interaction of N-BPs with FPPS in order to gain further insights into the mechanism of drug inhibition. Kinetic and crystallographic studies following site-directed mutagenesis of FPPS reveal key residues involved in stabilization of carbocation intermediate, substrate binding and formation of a tight enzyme-inhibitor complex. The aromatic ring of Tyr204 is involved in N-BP binding but not in the catalytic mechanism, where the hydroxyl moiety plays an important role. Lys200 is implicated in regulation of substrate binding, product specificity and enzyme isomerization which leads to a tight binding inhibition. Phe239 is considered important for the FPPS C-terminal switch which stabilizes substrate binding and promotes the inhibitor induced isomerized state. The highly conserved Arg112, Asp103 and Asp107 are pivotal for catalysis. Successful purification of the full length of Rab geranylgeranyl transferase (RGGT) complex downstream of the FPPS in the mevalonate pathway was achieved and may lead to co-crystallization with BP analogues and identification of the putative site of drug binding. Investigation of the in vitro effect of N-BPs on osteoclastogenesis suggest a correlation with FPPS inhibition kinetics for the most potent N-BPs but indicate an alternative mechanism of the disruption of bone resorption by alendronate. Together these results highlight the importance of the multiple interactions of N-BPs with side-chain residues of FPPS which dictate their strength of binding and advance the understanding of their pharmacophore effect.
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Welsche, Mathias Markus [Verfasser], and Roland [Akademischer Betreuer] Zengerle. "Determination of enzyme kinetics in micro concentration gradients = Bestimmung von Enzym-Kinetiken in Mikro-Konzentrationsgradienten." Freiburg : Universität, 2013. http://d-nb.info/1123475792/34.
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Ratnam, Kapila. "Spectroscopic and Kinetic Characterization of Oxomolybdenum Enzymes /." The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487934589977435.
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Diz, Filho Eduardo Britto dos Santos 1978. "Avaliação de anti-inflamatórios não esteroidais comerciais (AINES) sobre a atividade e função de PLA2 secretórias isolada dos venenos de Bothrops leucurus." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314476.
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Orientador: Marcos Hikari ToyamaTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Os Anti-inflamatórios não esteroidais (AINES) constituem uma classe de medicamentos com prescrição mundial, pois são fármacos que auxiliam na terapia de vários processos patológicos visto que oferecem ações farmacológicas: anti-inflamatória, analgésica e antipirética, como efeitos primários. A venda dos AINES no Brasil é de forma ''livre", porém, nos EUA é realizada somente sob prescrição médica. Apesar do grande número de informações acumuladas durante anos de pesquisa a respeito destas drogas sobre as atividades das Ciclooxigenases 1 e 2 (COX 1 e COX 2), respectivamente, do seu papel como ferramenta molecular para o estudo das vias biossintéticas das prostaglandinas e dos avanços em relação a sua síntese e síntese de novos AINES, não se tem conhecimento da atuação dessas drogas sobre a estrutura e função das Fosfolipases A2 secretórias (PLA2s), responsáveis pela produção do acido araquidônico precursor de vários tipos de derivados lipídicos biologicamente ativos e responsável pelo processo inflamatório. O objetivo deste trabalho é responder questões relacionadas à ação dos AINES sobre a ação de PLA2 de serpentes, responsáveis por eventos farmacológicos principalmente relacionados com edema. Esta atividade inflamatória pode ser desencadeada tanto por PLA2 enzimaticamente ativas, como não ativas, ambas induzindo o efeito de inflamação aguda por mecanismos ainda controversos. O trabalho utilizou PLA2 farmacologicamente ativa (destacadamente no processo inflamatório), isoladas do veneno de serpentes Bothrops leucurus, em ensaios de cinética enzimática, atividade farmacológicas relacionados ao edema, agregação plaquetária, mionecrose e neurotoxicidade conduzidos em presença das toxinas puras, das toxinas em presença dos AINES, em ensaios com prévio tratamento dos AINES e posterior aplicação das PLA2. Para uma avaliação mais precisa das alterações induzidas por estes AINES (princípios ativos puros ou drogas comerciais) foram empregadas técnicas de varredura em espectrômetro de UV-Vis (220-700mn) para averiguar possíveis modificações do espectro de absorbância da PLA2 e do AINES após a incubação o que poderia indicar modificações estruturais na PLA2, induzidas por estas drogas. Além de avaliar a ação específica dos AINES sobre as PLA2, secundariamente, o trabalho possibilitou conhecer alguns aspectos físico-químicos e estruturais de PLA2 isoladas do veneno de Bothrops leucuros. Os resultados determinaram que, embora não ocorram interações estruturais entre os AINES e as PLA2, foram evidenciadas alterações de atividade farmacológica
Abstract: Anti-inflammatory drugs (NSAIDs) are a class of prescription drugs worldwide, as are drugs that help in the therapy of various pathological processes as they offer pharmacological actions: anti-inflammatory, analgesic and antipyretic, such as primary effects. The sale of NSAIDs in Brazil are ''free", but the U.S. is preferred only by prescription. Despite the large amount of information accumulated through years of research on these drugs on the activities of the cyclooxygenases 1 and 2 (COX 1 and COX 2), respectively, of their role as a molecular tool for studying the biosynthetic pathways of prostaglandins and advances in respect of its synthesis and synthesis of new NSAIDs, there are no known performance of these drugs on the structure and function of secretory phospholipase A2 (PLA2s), responsible for the production of arachidonic acid precursor of various biologically active lipid derivatives and responsible for the inflammatory process. This paper aims to answer questions related to the action of NSAIDs on the action of PLA2 of snakes responsible for pharmacological events mainly related to edema. This inflammatory activity can be triggered by PLA2 enzymatically active and non active, inducing both the effect of acute inflammation by still controversial. The paper uses PLA2 pharmacologically active (highlighted in the inflammatory process), isolated from Bothrops leucurus snake venom and Bothrops jararacussu in kinetics assays, pharmacological activity related to edema, platelet aggregation, and neurotoxicity myonecrosis conducted in the presence of pure toxins, the toxins in the presence of NSAIDs, previous treatment trials with NSAIDs and the subsequent application of PLA2. For a more accurate assessment of changes induced by these NSAIDs (pure active ingredients or drugs trade) is employed techniques of scanning UV-Vis spectrophotometer (220-700nm) to ascertain possible modification of the absorbance spectrum of PLA2 and NSAIDs after incubation which could indicate structural changes in PLA2, induced by these drugs. In addition to assessing the specific action of NSAIDs on PLA2 secondarily work enables to know some physical-chemical aspects and structures of PLA2 isolated from the venom of Bothrops leucurus. The results determine that although there occur structural interactions between NSAIDs and PLA2 activity were observed pharmacological changes
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular