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1
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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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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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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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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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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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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Tenney, Joel David. "The kinetics of the chlorine dioxide generation reaction." Thesis, Georgia Institute of Technology, 1988. http://hdl.handle.net/1853/10020.
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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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Lai, Chung-Jeng. "Fumarate Activation and Kinetic Solvent Isotope Effects as Probes of the NAD-Malic Enzyme Reaction." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc278864/.
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The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.
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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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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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Fisher, Amanda Kaye. "Raman Chemometrics and Application to Enzyme Kinetics and Urinalysis." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/92590.
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Raman spectroscopy records the inelastic scattering of photons originating from striking a sample with monochromatic light. Inelastic, or Raman, scattered photons shift in wavelength due to excitation of the vibrational modes of molecules struck by the incident light. The Raman scattered photons are representative of all of the covalent bonds contained within a sample. Raman spectra taken of biological systems such as proteins, bacterial colonies, and liquid waste, are difficult to interpret due to the complexity of their covalent bond landscape and mixtures of molecules in highly variable concentrations. Rather than deconstructing Raman spectra to attempt assignment of specific bonds and functional groups to wavenumber peaks, here we have developed a chemometric analysis pipeline for quantifying the similarities and differences among a set of Raman spectra. This quantification aids in both classification of samples, and in measuring how samples change over time. The chemometric approach for interpretation of Raman spectra was made freely available in a user-friendly format via a MATLAB add-on called the Raman Data Analysis (RDA) Toolbox. Demonstrations of the RDA Toolbox functionalities on Raman spectra taken of various common biological systems are included, such as determination of protein concentration and monitoring bacterial culture growth. The RDA Toolbox and Raman spectroscopy are also used to initiate research in novel areas. Fast and accurate evaluation of enzyme specific activity is required for engineering enzymes, and results of Raman assays, evaluated in the RDA Toolbox, are successfully correlated to absorbance activity assays of an enzyme WT and mutant library. Further development of this research could alleviate the bottleneck of screening mutant libraries in enzyme engineering projects. The Toolbox is then used in a distinctly different application for evaluating urine and spent dialysate samples from patients with end stage renal disease. Categorization between samples from healthy volunteers and patients is accomplished with close to 100% accuracy, and evidence indicating that Raman spectroscopy can serve as an early diagnostic tool for infections of the peritoneal membrane is presented.PHD
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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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Cho, Yong Kweon. "Kinetic and Chemical Mechanism of Pyrophosphate-Dependent Phosphofructokinase." Thesis, University of North Texas, 1988. https://digital.library.unt.edu/ark:/67531/metadc332128/.
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Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the (βγ-bridge position of pyrophosphate to a (β-nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi. Neither back-exchange by [32p] nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction. Reduction of the pyridoxal 5'-phosphate-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 1,6-bisphosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphates probably directly coordinated to these phosphates. The pH dependence of kinetic parameters suggests that the enzyme catalyzes its reaction via general acid-base catalysis with the use of a proton shuttle. The base is required unprotonated in both reaction directions. In the direction of fructose 6-phosphate phosphorylation the base accepts a proton from the hydroxyl at C-l of F6P and then donates it to protonate the leaving phosphate. The maximum velocity of the reaction is pH independent in both reaction directions while V/K profiles exhibit pKs for binding groups (including enzyme and reactant functional groups) as well as pKs for enzyme catalytic groups. These data suggest that reactants bind only when correctly protonated and only to the correctly protonated form of the enzyme.
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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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Olsen, Greta A. "Characterization and modification of fluorogenic substrate coated particles for use as enzyme probes." Diss., Georgia Institute of Technology, 1999. http://hdl.handle.net/1853/27553.
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Pirinccioglu, Necmettin. "Modification of reactivity by supramolecular complex formation." Thesis, University of Kent, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309749.
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Morillas, Manuel. "Catalytic pathway and conformational stability of penicillin G acylase." Thesis, University of Newcastle Upon Tyne, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360276.
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Motiu, Stefan. "A theoretical study for the reactivation of O2 inhibited [Fe-Fe]-hydrogenase." Cleveland, Ohio : Cleveland State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1234364653.
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Thesis (Ph.D.)--Cleveland State University, 2008.Title from PDF t.p. (viewed on Apr. 6, 2009). Abstract. Includes bibliographical references (p. 99-109). Available online via the OhioLINK ETD Center. Also available in print.
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Dogaru, Daniela. "Hydrogenase inhibition by O2 density functional theory/molecular mechanics investigation /." Cleveland, Ohio : Cleveland State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1231721611.
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Thesis (Ph.D.)--Cleveland State University, 2008.Abstract. Title from PDF t.p. (viewed on Apr. 13, 2009). Includes bibliographical references (p. 102-109). Available online via the OhioLINK ETD Center. Also available in print.
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Barnes, John Ashley. "Theoretical modelling of transition states for chemical processes : application to enzymic and non-enzymic glycosidic hydrolysis." Thesis, University of Bath, 1994. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261066.
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Suharto, Adrian Rinaldi Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Structural studies of giardial arginine deiminase." Awarded by:University of New South Wales. Biotechnology and Biomolecular Sciences, 2006. http://handle.unsw.edu.au/1959.4/26293.
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Recombinant giardial arginine deiminase (rADI) was characterized. The enzyme was found to have a specific activity of 12 U (mg protein)-1under at pH 7.4 and 1 mM arginine. The maximum velocity was 14.75 U (mg protein-1) and the KM was 0.167 mM. The specific activity and maximum velocity values are significantly lower than the values reported previously for giardial rADI, while the KM value is quite similar. The optimum pH for the giardial rADI was 6-9, a broad range compared to other arginine deiminases. Recombinant ADI is very specific in its binding specificity, with canavanine (KI 2.4 mM) and ornithine (KI 2.1 mM) being the only substrate analogues giving significant inhibition from the wide variety of analogues tested. None of the analogues could be shown to act as alternative substrates. The contribution of conserved, catalytic and C-terminal residues proposed by previous research towards ADI activity was investigated by site-directed mutagenesis. Mutations of catalytic site residues Asp175, Glu226, His280 and Cys424 decreased the rADI activity to below 2%. Conservative mutations showed significant activity for Asp175 to Glu175 (DE175) and Glu226 to Asp226 (ED226). Site directed mutagenesis on the conserved non-catalytic site Leu46 showed activities below 15%, with the highest activity observed for the mutation to Val46 (LV46), which differs in one CH2 to Leu46 on its side chain. The KM of the mutant LV46 was 3.64 mM while for LA46 (Leu to Ala mutation) was 1.33 mM. Excising 5, 13, 16 amino acids from the C-terminal residues resulted in activity decreasing to 0.8% of the wild type, while excising 54 amino acids caused the rADI to be insoluble. Sequence alignment of Giardia and Dictyostelium suggests a homologous area within the C-terminal extension. Site directed mutagenesis on the Tyr567 residue in this region resulted in a decrease in activity, with the highest activity observed for a Tyr to Phe mutation. Studies using specific cysteine protease inhibitors demonstrated partial protection against proteolysis of ADI against giardial proteases. Degradation of ADI by giardial proteases was more rapid at pH 6 than at pH 7.4.
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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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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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Matosevic, S. "Design and characterisation of a prototype immobilised enzyme microreactor for the quantification of multi-step enzyme kinetics." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18918/.
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The large number of novel biocatalyst candidates available due to advances in protein engineering and evolution has driven research on automated microwell techniques for rapid catalyst evaluation and quantification of enzyme kinetics and stability. Interest in the further reduction in volume to the microfluidic scale has complemented these microwell approaches for the development of bioprocess operations due to their potential as inexpensive analytical tools with minute volumes and high throughput as well as for their potential for mass replication. This project involves the design and characterisation of a prototype immobilised enzyme microreactor (IEMR) on the inner surface of a 200 μm ID fused silica capillary. Immobilisation is achieved through affinity-based interaction between His6- tags engineered on the transketolase (TK) and transaminase (TAm) enzyme variants and Ni-NTA groups on the derivatised capillary surface. The microreactor concept was validated with two reactions, namely the transketolase-catalysed conversion of hydroxypyruvate (HPA) and glycolaldehyde (GA) to produce L-erythrulose followed by the conversion of erythrulose to 2−amino−1,3,4−butanetriol (erythrulose−aminotriol) in the presence of methybenzylamine (MBA) by CV (Chromobacterium violaceum)-derived ω- transaminase. These keto- and aminodiol synthons are synthetically very useful in the production of a range of compounds with pharmaceutical application. The principles of stop-flow (batch) kinetics were initially investigated with respect to the catalytic performance of both enzymes, where the reaction was shown to depend on substrate concentration and residence time. TK kinetic parameters, evaluated based on a Michaelis−Menten model, in the IEMR (Vmax(app) = 0.1 ± 0.02 mmol.min-1, Km(app) = 26 ± 4 mM) were shown to be comparable to those measured in free solution. Furthermore, the kcat for the microreactor of 2.1 s−1 was similar to the value of 3.9 s−1 for the bioconversion in free solution. This was attributed to the controlled orientation and monolayer surface coverage of the His6−immobilised TK. Furthermore the quantitative elution of the immobilised TK and the regeneration and reuse of the derivatised capillary over 5 cycles were also demonstrated. Whilst slower than TK, the TAm reaction in the IEMR showed similar catalytic performance to a standard reaction in glass vials. Stopped−flow bioconversion results were complemented by continuous flow kinetics of the TK reaction with on-line UV detection (ActiPix, Paraytec), where the dependence of reaction kinetics on flow conditions was investigated. The Km(app), evaluated based on a continuous flow kinetic model, was shown to increase with flow rate, with the optimal being at the lowest flow rates used (0.2 μL.min-1). Furthermore, the value of Km(app) was shown to approach the value of the Michaelis constant of the free enzyme under zero flow (∼25 mM). The prototype microfluidic system was then implemented for the quantitative evaluation of multi-step TK-TAm bioconversion kinetics and the formation of chiral amino diol 2-amino-1,3,4- butanetriol (ABT) product from achiral substrates was demonstrated. The rate of accumulation of ABT (also referred to as EAT) by TAm was 0.02 mM.min-1.μgTAm -1, which was 4× slower than the rate of the TK−catalysed step. Demonstration of the synthesis of the product via the dual reaction and the monitoring of each component provided a full profile of the little known bioconversion and demonstrated the potential for creating novel multi−enzyme pathways in lab−on−a−chip systems, which further enables multi-substrate screening and screening of libraries of evolved enzymes of interest to be achieved rapidly and economically. This in vitro study of multi-step enzyme kinetics provides insight into the behaviour of these de novo engineered pathways to aid incorporation into suitable host cells.
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Mallick, Sushanta. "Kinetic mechanism of NAD-malic enzyme from Ascaris suum in the direction of reductive carboxylation of pyruvate." Thesis, University of North Texas, 1990. https://digital.library.unt.edu/ark:/67531/metadc332658/.
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For this pseudoquadreactant enzymatic reaction (Mn2+ is a psuedoreactant), initial velocity patterns were obtained under conditions in which two substrates were maintained saturating while one reactant was varied at several fixed concentrations of the other.
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Jones, J. "Kinetic studies on NADsup(+):ADPribosyltransferase." Thesis, University of Reading, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372662.
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Hau, Stephen S. "Frequency-domain enzyme kinetics in the context of artificial calcium oscillations." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/39073.
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Avis, Johanna M. "Studies on tRNA charging by tyrosyl-tRNA synthetase." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239594.
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Yelloly, Julia M. "Ecological role of surface phosphatase activities of Rivulariaceae." Thesis, Durham University, 1996. http://etheses.dur.ac.uk/5086/.
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The literature suggests that the cyanobacterium Rivularia is found at sites where organic phosphorus (P), at times forms a high proportion of total P in the environment. Its ability to utilise organic P through "surface" phosphatase activity may be important in its success. The aim of this thesis was to investigate this in detail using both field material (from a freshwater stream and from a marine intertidal zone) and axenic isolates of Rivulariaceae. At both sites inorganic P concentrations peaked: in March/May (1992-4) at the freshwater site, and in June (1992-3) at the marine site (although in 5 of the pools containing Rivularia, inorganic P was maximal in February/March 1993). Pools associated with rotting seaweed had higher concentrations of inorganic P (which made up most of the total P) during peaks. It is likely that the high tide resulted in the mixing of weed pool water with Rivularia pool water, slightly lower down the eulittoral zone, and also influenced the retreating seawater. Organic P was a greater proportion of total P in the pools containing Rivularia and was found to increase in these pools during the tidal cycle, suggesting internal generation. At the freshwater site organic P concentrations were higher in pools associated with peat than in stream water. At the freshwater site phosphorus fractions were often below detection limits, but combined nitrogen was rarely this low; the reverse was the case at the marine site. At the freshwater site phosphomonoesterase activity of Rivularia was generally high, except when hormogonia were present in the colonies. At the marine site, phosphatase activity was usually low, with a peak using p-nitrophenyl phosphate (pNPP) as a substrate in July/August and, using 4-methyIumbelliferyl phosphate (4-MUP) as a substrate in September/October, especially in 1992. Phosphatase activity of Rivularia at both sites was negatively influenced by inorganic P and positively correlated with the presence of hairs. K(_m) (Michaelis-Menten constant) was lower using 4-MUP than pNPP in all organisms assayed. Apparent negative cooperativity was found in 7-day cultures of Calothrix parietina D550 using 4-MUP and in whole colonies of freshwater Rivularia using pNPP. These results were discussed with reference to the relationship between nitrogen (N) and P at the sites and the enzyme kinetics of field organisms and axenic isolates. Rivularia is successful in these apparently different environments probably because organic P can be utilised when phosphorus is limiting and colonies are able to fix N2 when N is limiting. Seasonal peaks in inorganic P probably allow regeneration of the Rivularia population.
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Davies, Elizabeth. "The effects of pulsed magnetic fields on the adenylate cyclase enzyme system in aggregating Dictyostelium discoideum." Thesis, University of Brighton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239721.
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Howard, Bruce Riley. "The crystal structure of malate synthase and mechanistic implications /." view abstract or download file of text, 1999. http://wwwlib.umi.com/cr/uoregon/fullcit?p9948022.
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Thesis (Ph. D.)--University of Oregon, 1999.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 67-71). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9948022.
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Borda, Emily J. "Investigation of ribozyme structure and dynamics through photochemical crosslinking and metal ion cleavage /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/11616.
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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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Lin, Man Guang 1966. "Continuous flow microwave heating : evaluation of system efficiency and enzyme inactivation kinetics." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82281.
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A continuous flow microwave heating system was set up by using one domestic microwave oven (1000W nominal output at 2450MHz). Water was run through the coil centrally located inside the oven cavity for microwave heating. Microwave absorption efficiency was evaluated by measuring inlet and outlet temperatures of coil as a function of system variables. In order to optimize the coil configuration, the influence of tube diameter (6.4, 7.9 and 9.7mm); initial temperature (10, 20 and 30 ºC); number of turns (3.5, 4.5 and 5.5); coil diameter (61.5, 88, 102, 121 and 153 mm) and pitch (16, 18, 20, 22 and 24mm) were evaluated, respectively at different flow rates (240, 270, 300, 330 and 360ml/min). In helical systems, Dean number is used as a measure of secondary flow which enhances mixing of the fluid providing uniform heating even under laminar flow conditions. Results showed that microwave absorption efficiency was a compromise between coil volume and Dean number. Therefore, a helical coil (110 mm high) with a coil diameter of 108 mm, tube diameter of 8.2 mm, 5.5 turns demonstrated the highest efficiency, fast heating rate, more uniform heating and less temperature fluctuations. The optimized coil configuration parameters were used subsequently to set up continuous-flow microwave heating system.
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Rostami-Hodjegan, Amin. "Kinetics of metabolites : implications in the assessment of bioequivalence and enzyme activity." Thesis, University of Sheffield, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245578.
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Mehta, Bhavya Chandrakant. "Optimization of enzyme dissociation process based on reaction diffusion model to predict time of tissue digestion." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1142575553.
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Scotney, Pierre David. "The catalytic mechanism of Bacillus stearothermophilus pyruvate kinase." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266959.
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Downey, Aaron. "Synthesis and MAO activity of a series of benzimidazolyl and indazolyl prodrugs." Thesis, Virginia Tech, 2006. http://hdl.handle.net/10919/35612.
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Parkinson's disease (PD) is a chronic, progressive disorder of the central nervous system that affects approximately 1.5 million Americans. One of the principal pathological features of PD is dopamine deficiency in the substantia nigra of the brain. A key enzyme that has been associated with the neurodegeneration seen in PD is monoamine oxidase-B (MAO-B). Several inhibitors of this enzyme have resulted in neuroprotection in the mouse model of PD. One such compound is 7-nitroindazole (1).
This thesis describes the synthesis and MAO activity of several indazolyl and benzimidazolyl prodrugs that are designed to release an enzyme inhibitor in the affected brain area. These studies have provided information regarding the nucleophilic aromatic substitutions of the ambident nucleophiles under consideration. We have also discovered a compound that releases the enzyme inhibitor upon bioactivation by MAO. These results as well as a MPTP mouse study with the aforementioned compound are detailed within.Master of Science
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Jennings, Natalie A. "Acetylcholinesterase in the sea urchin Lytechinus variegatus characterization and developmental expression in larvae /." Birmingham, AL : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2008r/jennings.pdf.
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Collett, Michael. "An allosteric network within dynamin." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/15871.
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Dynamins are large enzymes that catalyse the hydrolysis of GTP (GTPase activity). They assemble through oligomerisation into helical polymers during endocytosis to facilitate membrane scission and vesicle release in all known eukaryotic cell types. Dynamin oligomerisation is associated with an increase in GTPase activity. This drives constriction of the dynamin polymer to induce membrane fission at the neck of endocytic vesicles, releasing the new vesicles into the cytoplasm. This requires GTP hydrolysis at multiple G domains within the helix to be coordinated. The underlying mechanisms that regulate such coordination remain poorly understood. It is reasonable to predict that these enzymes possess a highly developed allosteric network that is responsible for this coordination. There are three classical dynamin (dynI-III) genes in the mammalian genome, each of which is alternately spliced. DynI is a neuronal isoform required for synaptic vesicle recovery mechanisms within the presynapse of neurons. There are 5 dynamin domains: the catalytic G domain; the bundle signalling element (BSE); the dynamin middle domain; the pleckstrin homology (PH) domain; and the C-terminal proline rich domain (PRD). Dyn I and II share a homologous splice site within the middle domain, which is the primary domain mediating assembly. This splice site produces two variants ‘a’ and ‘b’ with the alternate exon encoding the complete second α-helix of the middle domain (Mα2). This helix is part of a predicted regulatory interface in dynamin that allosterically regulates the G domain via the BSE The overall aim was to understand dynamin allostery by focusing on the predicted regulatory interface between the middle domain α2 helix (Mα2) and the BSE of dynamin, which is known as interface 5. The work utilised natural splice variation of the Mα2 as a key tool to explore the properties of this interface and to uncover the role of alternative splicing at this site. Firstly, it was hypothesised that alternate splicing of Mα2 in dynI would produce variants with unique assembly and activity properties. This was addressed by characterising these properties using recombinantly expressed and purified dynamin splice variants. A combination of activity assays and biophysical techniques were used to compare the two dynI isoforms. The data suggested fundamental differences in the activity and assembly of these isoforms. Cell-based endocytosis assays confirmed they«br /» have different endocytic capacities. Quantitation of the level of each isoform in the developing rat brain revealed a remarkable isoform-specific regulation of protein expression, with the dynIa isoform greatly increasing in abundance with the formation of synaptic terminals while the dynIb isoform changed little during development. This supports our hypothesis that dynamin middle domain splice variation produces isoforms that exhibit modified enzyme properties and further suggests that these represent adaptations to specific cellular functions. Second, the relationship between the Mα2 helix and the G domain was explored. This analysis began with direct comparison of previously published crystal structures of each isoform, and a distinct rearrangement of key catalytic loops within the G domain was identified. This predicted changes in the GTP binding affinity of the isoforms, which was experimentally validated using substrate kinetics under a variety of assembly conditions. The data confirmed the predictions made from structural comparison. It also revealed for the first time the presence of allosteric coupling of substrate binding between G domains, which we call 'G domain coupling'. This represents a novel finding for any enzyme within the dynamin family. G domain coupling was responsive to assembly state, a phenomenon we called the 'dynI allosteric switch'. This causes the coupling of G domains to be activated or inactivated according to assembly state. The conditions that activate G domain coupling were different for the dynI isoforms, with coupling activated in the basal state for dynIb, and the helical state for dynIa. This represents the biggest functional difference between these two isoforms. Third, a combination of computational and mutagenesis studies enabled identification of residues that may contribute to allosteric communication within dynamin. Allostery was mediated by an extensive allosteric network that functionally couples the G domain to the PH domain, PRD and middle domains. By analysing the hydrolysis activity of a variety of dynamin isoforms and mutants, the Mα2 helix was identified as a key component of this network via the intermolecular interface 5, which exists between Mα2 and a neighbouring BSE domain within the tetramer building block. The data revealed the existence of an overall assembly signal propagation pathway within both unassembled dynamin tetramers and within oligomers that differentially regulates GTP hydrolysis according to the assembly state, via interface 5. The overall work represents a fundamentally new understanding of the catalytic mechanism of dynamin enzymes. The study is the first to systematically reveal and characterise an allosteric network operating within the classical dynamins. This network regulates a previously uncharacterised form of allostery between dynamin G domains. It therefore has important implications for the coordination of GTP hydrolysis within the helix and therefore, the vesicle scission mechanism of endocytosis. Elements of this network are conserved within the classical dynamins, and this may represent conservation within the broader dynamin family.
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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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Yu, Peng. "Allosteric regulation of glycerol kinase: fluorescence and kinetics studies." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/1537.
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Glycerol kinase (GK) from Escherichia coli is allosterically controlled by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier protein IIAGlc of the phosphotransferase system. These controls allow glucose to regulate glycerol utilization. Fluorescence spectroscopic and enzyme kinetic methods are applied to investigate these allosteric controls in this study. The linkage between FBP binding and GK tetramer assembly is solved by observation of homo-fluorescence energy transfer of the fluorophore Oregon Green (OG) attached specifically to an engineered surface cysteine in GK. FBP binds to tetramer GK with an affinity 4000-fold higher than to dimeric GK. A region named the coupling locus that plays essential roles in the allosteric signal transmission from the IIAGlc binding site to the active site was identified in GK. The relationship between the coupling locus sequence in Escherichia coli or Haemophilus influenzae GK variants and the local flexibility of the IIAGlc binding site is established by fluorescence anisotropy determinations of the OG attached to the engineered surface cysteine in each variant. The local flexibility of the IIAGlc binding site is influenced by the coupling locus sequence, and in turn affects the binding affinity for IIAGlc. Furthermore, the local dynamics of each residue in the IIAGlc binding site of GK is studied systematically by the fluorescence anisotropy measurements of OG individually attached to each position of the IIAGlc binding site. The fluorescence steady-state anisotropy measurement provides a valid estimation of the local flexibility and correlates well with the crystallographic B-factors. Steady-state kinetics of FBP inhibition shows that the data are best described by a model in which the partial inhibition and FBP binding stoichiometry are taken into account. Kinetic viscosity effects show that the product-release step is not the purely rate-limiting step in the GK-catalyzed reaction. Viscosity effects on FBP inhibition are also discussed.
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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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Hamberg, Anders. "Serine Hydrolase Selectivity : Kinetics and applications in organic and analytical chemistry." Doctoral thesis, KTH, Biokemi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-12831.
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The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved. In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase. In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches. In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance. In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.QC20100629
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Apperley, Kim Yang-Ping. "Reversible and Photolabile Inhibitors for Human Tissue Transglutaminase." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36593.
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Tissue transglutaminase (TG2) is a calcium-dependent enzyme that natively catalyses the formation of isopeptidic bonds between protein- or peptide-bound glutamine and lysine residues. Physiologically, it is ubiquitously expressed in tissues, with roles in cellular differentiation, extracellular matrix stabilisation, and apoptosis, among others. However, its unregulated activity has been associated with various pathologies including fibrosis, cancer and celiac disease.
Since most pathologies are associated with an increased transamidation activity, efforts have been directed towards the development of TG2 inhibitors. In this context, the work described in this thesis is centred on reversible inhibitors, building on recent work done within the Keillor group in two directions, namely localisation and potency.
In a localisation-driven approach, we developed a photolabile derivative of a known reversible inhibitor, in order to form a covalent bond with the enzyme and determine the inhibitor’s binding site. In tandem, we optimised a protocol for the expression of TG2 incorporating ArgΔ10 and LysΔ8, amino acids that are 13C- and 15N-labelled to provide a mass shift of 10 and 8 Da, respectively, compared to the corresponding unlabelled amino acids. This “heavy” TG2 was developed as a tool for reference in the analysis of the tryptic digest of labelled protein.
In a potency-driven approach, based on the observation that previous trans cinnamoyl inhibitor scaffolds were susceptible to nucleophilic attack by glutathione, we developed a bis(triazole) scaffold with reduced electrophilicity. The preparation of a small library of compounds showed that this scaffold demonstrates a preference for electron-withdrawing substituents, such as nitro groups.
Continuing in a potency-driven approach, and inspired by work done in the identification of glutathione-resistant scaffolds, we studied a new alkynyl scaffold. While still susceptible to glutathione addition, these compounds showed a marked improvement in potency, with the lead compound having an IC50 of 930 nM and being established as a competitive inhibitor with a Ki of 420 nM, our most potent reversible inhibitor to date. Furthermore, this scaffold also produced an inhibitor lacking nitro groups (to limit eventual cellular toxicity), but maintaining good potency, with an IC50 value of 3.03 μM.
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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)