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1
Lu, Siran. "Single molecule kinetic isotope effect." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526483.
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Kopec-Harding, Kamilla Rosa. "Computational studies of the kinetic isotope effect inmethylamine dehydrogenase." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/computational-studies-of-the-kinetic-isotope-effect-inmethylamine-dehydrogenase(b6883173-40ea-4a35-948b-c966105230cd).html.
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There is currently experimental evidence of hydrogen tunnelling in over 20 different enzymes include yeast alcohol dehydrogenase (YADH), morphinone reductase (MR) and methylamine dehydrogenase (MADH). Various models have been used to describe hydrogen tunnelling in enzymes including the static barrier model, the vibrationally enhanced ground state tunnelling model (VEGST) and the environmentally coupled tunnelling model (ECT). Despite some differences in these models, there is a general consensus that a temperature dependent kinetic isotope effect (KIE) is indicative of tunnelling dominated by a ratepromoting motion. Stopped flow studies of MADH with ethanolamine as substrate (mm-MADH/EA) show that the KIE of the proton transfer decreases with temperature - within the framework of the ECT model, the kinetics of this proton transfer are consistent with ground state tunnelling dominated by active dynamics (a promoting vibration). However, an alternative hypothesis is that this temperature dependence can be attributed to the population of multiple reactive configurations within the active site. If distinct substrate configurations are associated with distinct kinetic behaviour, the temperature dependence of the KIE could be due to temperature dependent fluctuations in the relative populations of these configurations. Long and short time molecular dynamics simulations of mm-MADH/EA were carried out to explore both of these scenarios. Theethanoliminoquinone intermediate was found to adopt a number of different hydrogen bonding configurations in the active site of MADH. Adiabatic scans of the proton transfer event in conjunction with WKB calculations of the KIE showed that these hydrogen bonding patterns are associated with different barrier heights and KIEs. However, simple modelling with the Boltzmann distribution showed that fluctuations in the relative population of these configurations of the magnitude expected in the temperature range 278K-308K leads to negligible changes in the magnitude of the KIE. This suggested thatmultiple reactive configurations are unlikely to account for the temperature dependence of the KIE. Spectral density analysis of the short-time MD simulations was then carried out try to identify any promoting motions in mm-MADH/EA. Since no evidence of promoting motions was found, the origin of the temperature dependence on the KIE remains an open question: the analysis in this study was restricted to one of four possible proton transfers in this substrate (HI3-OD1). Further work might look at the possibility of a promoting motion pertinent to the other transfers.
3
Burke, Erin E. "Heavy atom and hydrogen kinetic isotope effect studies on recombinant, mammalian sialyltransferases." [Gainesville, Fla.] : University of Florida, 2005. http://purl.fcla.edu/fcla/etd/UFE0011586.
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Ingle, Shakti Singh. "RNA structure investigation: a deuterium kinetic isotope effect/hydroxyl radical cleavage experiment." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12787.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.The hydroxyl radical is widely used as a high-resolution footprinting agent for DNA and RNA. The hydroxyl radical abstracts a hydrogen atom from the sugar- phosphate backbone of a nucleic acid molecule, creating a sugar-based radical that eventually results in a strand break. It was shown previously that replacement of deoxyribose hydrogen atoms with deuterium results in a kinetic isotope effect (KIE) on hydroxyl radical cleavage of DNA. The KIE correlates well with the solvent accessible surface area of a deoxyribose hydrogen atom in DNA. We chose the structurally well-defmed sarcin-ricin loop (SRL) RNA molecule as a model system to extend the deuterium KIE/hydroxyl radical cleavage experiment to RNA. We observed a substantial KIE upon deuteration of the 5'-carbon of the ribose. Values ranged from 1.20 to 1.96, and depended on the position of the residue within the SRL. We found a smaller KIE upon 4'-deuteration. Values ranged from 1.05 to 1.23. Values of 5' and 4' KIEs correlate with the extent of cleavage and with the solvent accessible surface areas of ribose hydrogen atoms ofthe SRL. Gel electrophoresis of cleavage products reveals that the strand break is terminated at the 5' end by multiple chemical species. Upon 3'-radiolabeling a specifically 5'-deuterated SRL RNA molecule, we observed a KIE on the production of a cleavage product having a gel mobility different from that of a phosphate-terminated RNA strand. Reduction with sodium borohydride gave rise to an RNA fragment terminated by a 5'-hydroxyl group. These experiments are consistent with 5' hydrogen abstraction by the hydroxyl radical producing a 5'-aldehyde-terminated RNA strand that retains the nucleotide from which the hydrogen atom was abstracted. This is the first report of such a species. This chemistry has important implications for the interpretation of structural analysis experiments on RNA that rely on primer extension to synthesize eDNA copies of hydroxyl radical cleavage products. The different 5'-terminated products resulting from hydroxyl radical cleavage at a given nucleotide would yield cDNAs of two different lengths, thereby distributing the cleavage intensity over two nucleotides instead ofone and lowering the resolution ofthe experiment.
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Yousefi-Shivyari, Niloofar. "Isotope ratios in source determination of formaldehyde emissions." Thesis, Virginia Tech, 2020. http://hdl.handle.net/10919/99308.
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Formaldehyde emissions from non-structural wood composites are regulated and the regulation target is urea-formaldehyde (UF) resin. UF resins are hydrolytically unstable and constantly emit formaldehyde as a function of temperature and relative humidity. When heated, wood also generates formaldehyde, but this was of little concern until 2010 when formaldehyde regulations became much more demanding. This regulation motivated the industry to account for all formaldehyde sources, synthetic as from resin, and biogenic as from wood. This effort represents first steps towards quantifying biogenic and synthetic contributions to formaldehyde emissions in non-structural wood composites.
It is possible to distinguish the 13C/12C isotope ratio of UF resins from the isotope ratio of plant biomass. Conditions during and after composite hot-pressing promote reactions that generate formaldehyde from wood and UF resin, and the kinetic isotope effect continuously lowers the product isotope ratios as a function of yield. If such isotope fractionation did not occur, it would be a simple matter to quantify contributions of wood and UF resin to formaldehyde emissions using static isotope ratios. Isotope fractionation, therefore, complicates the requirements for distinguishing biogenic and synthetic formaldehyde in wood composite emissions. Those requirements are 1) establish the reference carbon isotope ratios in wood and in UF resin (just the formaldehyde portion of UF), and 2) estimate the kinetic isotope effects in formaldehyde generation by wood and cured UF resin. The latter requirement fixes a range for the respective isotope ratios; the numerical ranges enable a simple model of the average isotope ratio for a mixture of biogenic and synthetic formaldehyde in wood composite emissions. Finally, the measured isotope ratio of captured emissions would be compared to the model.
This work did not achieve all aspects of the requirements mentioned, but a solid foundation was established for future completion of the ultimate goals. In reference to requirement 1, the carbon isotope ratio of experimental Pinus taeda wood was accurately measured (including some isolated fractions) using isotope ratio mass spectroscopy (IRMS). IRMS of UF resin first requires removal of urea carbons- UF resin was subjected to acid hydrolysis and capture of the resin formaldehyde into aqueous ammonium hydroxide. This provided a nearly quantitative conversion (negligible isotope fractionation) of resin formaldehyde into hexamine for IRMS. Using this hexamine method, the formaldehyde carbon isotope ratios of two industrial UF resins were accurately measured, demonstrating basic feasibility for the project goal.
Estimating the kinetic isotope effect (Requirement 2) required creation of a thermochemical reactor, where wood or cured UF resin was heated under N2 flow such that the emitted formaldehyde was easily captured. In this case, conversion of captured formaldehyde into hexamine was abandoned in favor of silica gel cartridges loaded with sodium bisulfite. Isolation and IRMS of the formaldehyde-bisulfite adduct were effective and considered easily transferable to industrial settings. This system was employed to measure fractionation in cured resin as a function of relative humidity, and in Pinus taeda wood as a function of relative humidity, temperature, and time. More information about isotope fractionation is required; but most notable was the fractionation behavior in wood where evidence was found for multiple formaldehyde generating reactions. Overall, this work established feasibility for the goals and laid the foundation for future efforts.Master of Science
Home-interior products like cabinetry are often produced with wood composites adhesively bonded with urea-formaldehyde (UF) resin. UF resins are low cost and highly effective, but their chemical nature results in formaldehyde emission from the composite. High emissions are avoided, and the federal government has regulated and steadily reduced allowable emissions since 1985. The industry continuously improved UF technologies to meet regulations, as in 2010 when the most demanding regulations were implemented. At that time, many people were unaware that wood also generates formaldehyde; this occurs at very low levels but heating during composite manufacture causes a temporary burst of natural formaldehyde. Some wood types produce unusually high formaldehyde levels, making regulation compliance more difficult. This situation, and the desire to raise public awareness, created a major industrial goal: determine how much formaldehyde emission originates from the resin and how much originates from the wood. These formaldehyde sources can be distinguished by measuring the carbon isotope ratio, 13C/12C. This ratio changes and varies due to the kinetic isotope effect. Slight differences in 13C and 12C reactivity reveal the source as either petrochemical (synthetic formaldehyde) or plant-based (biogenic formaldehyde). This work demonstrates that achieving the industry goal is entirely feasible, and it provides the analytical foundation. The technical strategy is: 1) establish reference isotope ratios in wood and in UF resin, and 2) from the corresponding wood composite, capture formaldehyde emissions, measure the isotope ratio, and simply calculate the percentage contributions from the reference sources. However, a complication exists. When the reference sources generate formaldehyde, the respective isotope ratios change systematically in a process called isotope fractionation (another term for the kinetic isotope effect). Consequently, this effort developed methods to measure fractionation when cured UF resin and wood separately generate formaldehyde, with greater emphasis on wood. Isotope fractionation in wood revealed multiple fractionation mechanisms. This complexity presents intriguing possibilities for new perspectives on formaldehyde emission from wood and cured UF resin. In summary, this work demonstrated how source contributions to formaldehyde emissions can be determined; it established effective methods required to refine and perfect the approach, and it revealed that isotope fractionation could serve as an entirely novel tool in the materials science of wood composites.
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MacMillar, Susanna. "Isotopes as Mechanism Spies : Nucleophilic Bimolecular Substitution and Monoamine Oxidase B Catalysed Amine Oxidation Probed with Heavy Atom Kinetic Isotope Effects." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis (AUU), 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7441.
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Pagano, Philip Lee Jr. "Investigating fast dynamics at the tunneling ready state in formate dehydrogenase." Diss., University of Iowa, 2017. https://ir.uiowa.edu/etd/5592.
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Enzyme dynamics occur on a wide range of length and timescales. This work is focused on understanding enzyme dynamic at the fs-ps timescale as this is the dynamic range at which bonds are typically made and broken during chemical reactions. Our work focuses on enzymes that catalyze hydride transfer between two carbon atoms - a fundamental reaction in biology. Primary kinetic isotope effects and their temperature dependence have implied that fast dynamics of the enzyme are important in facilitating hydride transfer, however these experiments do not measure any such motions directly. We make use of two-dimensional infrared spectroscopy (2D IR), a technique that interrogates the vibrations of molecules to extract dynamic information from the surrounding environment with 100 fs resolution. A model system, formate dehydrogenase (FDH), is an excellent probe of dynamics at the fs-ps timescale. Azide bound to the ternary complex of FDH offers the ability to measure dynamics of an analog structure of the reactive complex using 2D IR, while also studying the reaction directly with and KIE’s and their temperature dependence. By altering various parts of the structure of FDH via mutagenesis and other techniques, we investigate the role of structure and dynamics to determine how fast dynamics of the active site influence the the kinetics of hydride transfer. These experiments are the first means of providing a dynamic interpretation of KIEs and their temperature dependence.
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Indurugalla, Deepani. "A kinetic isotope effect study on the acid-catalyzed hydrolysis of methyl xylopyranosides and methyl 5-thioxylopyranosides." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0021/NQ37716.pdf.
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Lorenzini, Leonardo. "Effects of T3 and 3-iodothyronamine (T1AM) on cellular metabolism, and influence of serum proteins on T1AM assay." Doctoral thesis, Università di Siena, 2018. http://hdl.handle.net/11365/1046523.
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Thyroxine (T4) is the predominant form of thyroid hormone (TH). In target tissues, T4 is enzymatically deiodinated to 3,5,3′-triiodothyronine (T3), a high-affinity ligand for the nuclear TH receptors TRα and Trβ. T3 modulates genes transcription via activation of TRα and TRβ. Non-genomic effects have also been described.
In 2004 the research groups of professors Scanlan Grandy and Zucchi discovered an endogenous thyroid hormone derivative called 3-iodothyronamine (T1AM). They proved that at nanomolar concentrations it can activate trace amine associated receptors 1 (TAARs)[1] and it may also interact with other targets, such as plasma membrane transporters, mitochondrial proteins and vesicular biogenic amine transporters [2]–[4]. Endogenous T1AM has been detected in human and rodent blood and tissues samples by liquid chromatography coupled mass spectrometry (LC/MS/MS) [5]. Its endogenous levels are a matter of argument due to the challenges that its accurate quantification poses. For this reason, so far a worldwide adopted extraction method has not been established. Circulating T1AM has so far been considered to be largely bound to apolipoprotein apoB100 [6].
The first T1AM functional effect to be discovered was severe hypothermia [7]. This effect is associated to a decrease in oxygen consumption and a reduction of the respiratory quotient (CO2/O2), which reflects the relationship between glucose and fatty acid oxidation, resulting in a shift from carbohydrate to lipid as energy source [8]. The molecular mechanisms underlying T1AM effects are still unknown, however Mariotti and colleagues [9] analyzed gene expression profiles in adipose tissue and liver of T1AM chronically treated rats and found significant transcriptional effects involving sirtuin genes, which regulate important metabolic pathways.
Therefore, the first aim of this work was to compare the effect of T1AM and T3 chronic treatment on mammalian sirtuin expression in hepatoma cells (HepG2) and isolated hepatocytes.
Isolated rat hepatocytes were obtained by liver in-situ collagenase perfusion. Sirtuin expression was determined by Western Blot analysis in cells treated for 24 h with 1-20 µM T1AM or T3. In addition, cell viability was evaluated by MTT test upon 24 h treatment with 100 nM to 20 µM T1AM or T3. In HepG2, T1AM significantly reduced SIRT1 and SIRT4 protein expression at 20 µM while T3 strongly decreased the expression of SIRT1 (20 µM) and SIRT2 (any tested concentration). In primary rat hepatocytes T1AM, did not affect protein expression whereas T3 decreased SIRT2 at 10 µM. The extent of MTT-staining was moderately but significantly reduced by T1AM, particularly in HepG2 cells, in which the effect occurred at concentration starting from 100 nM. T3 reduced MTT staining in HepG2 but not in isolated hepatocytes. T1AM and T3 differently affected sirtuin expression in hepatocytes. Since SIRT4 is an important regulator of lipid and glucose metabolism, whereas SIRT1 and SIRT2 have a key role in regulating cell cycle and tumorigenesis, our observations are consistent with the shift from carbohydrates to lipids induced by T1AM and indicate a potential new role of T1AM in modulating tumor proliferation.
The second part of this project was aimed at clarifying the issue that, so far, every research group working on this molecule has encountered when trying to accurately quantifying T1AM endogenous levels. These difficulties were usually attributed to problems in extraction or other pre‐analytical steps. Most researchers have developed various workaround for this issue. For example, on cell culture experiments, to avoid the presence of serum proteins in the culturing media, experiments have often be performed with unphysiological protein‐free media.
The second goal of this project was therefore to evaluate the effect of serum protein on the recovery of exogenous T1AM.
Cell culture media (Krebs buffer, DMEM, FBS, DMEM+FBS, used either in the absence or in the presence of NG108‐15 cells) and other biological matrices (rat brain and liver homogenates, human plasma and blood) were spiked with T1AM and/or deuterated T1AM (d4‐T1AM) and incubated for times ranging from 0 to 240 min. Samples were extracted using a liquid/liquid method and analysed using liquid chromatography coupled to mass spectrometry (LC-MS/MS), to assay T1AM and some of its metabolites. For the first time in the history of this molecule, in FBS‐containing buffers, an exponential decrease in T1AM levels was observed over time. T1AM metabolites were not detected, except for minimum amounts of TA1. Notably, d4‐T1AM decreased over time at a much lower rate, reaching 50‐70% of the baseline at 60 min. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide, however, the process could not be reverted. In the presence of cells, T1AM concentration decreased virtually to 0 within 60 min, but TA1 accumulated in the incubation medium, with quantitative recovery. Spontaneous decrease in T1AM concentration with isotopic difference was confirmed in rat organ homogenates and human whole blood. Conclusions. On the whole, these results suggest binding and sequestration of T1AM by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenousT1AM.
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Richan, Teisha. "Conservative Tryptophan Mutations in Protein Tyrosine Phosphatase PTP1B and its Effect on Catalytic Rate and Chemical Reaction." DigitalCommons@USU, 2017. https://digitalcommons.usu.edu/etd/5584.
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Protein-tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosphorylated tyrosines by a 2-step mechanism involving nucleophilic attack by cysteine and general acid catalysis by aspartic acid. In most PTPs the aspartic acid resides on a flexible protein loop, consisting of about a dozen residues, called the WPD loop. PTP catalysis rates span several orders of magnitude, and differences in WPD loop dynamics have recently been show to correlate with the rate of enzymatic catalysis. The rate of WPD loop motion could possibly be related to a widely conserved tryptophan residue on the WPD loop. Therefore, point mutants were made in PTP1B (a human PTP) to the conserved tryptophan residue and their effects on catalytic rate and chemical reaction were studied. The results of these studies are presented in this thesis.
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Giebel, Brian M. "Advancement and Application of Gas Chromatography Isotope Ratio Mass Spectrometry Techniques for Atmospheric Trace Gas Analysis." Scholarly Repository, 2011. http://scholarlyrepository.miami.edu/oa_dissertations/610.
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The use of gas chromatography isotope ratio mass spectrometry (GC-IRMS) for compound specific stable isotope analysis is an underutilized technique because of the complexity of the instrumentation and high analytical costs. However stable isotopic data, when coupled with concentration measurements, can provide additional information on a compounds production, transformation, loss, and cycling within the biosphere and atmosphere. A GC-IRMS system was developed to accurately and precisely measure δ13C values for numerous oxygenated volatile organic compounds (OVOCs) having natural and anthropogenic sources. The OVOCs include methanol, ethanol, acetone, methyl ethyl ketone, 2-pentanone, and 3-pentanone. Guided by the requirements for analysis of trace components in air, the GC-IRMS system was developed with the goals of increasing sensitivity, reducing dead-volume and peak band broadening, optimizing combustion and water removal, and decreasing the split ratio to the IRMS. The technique relied on a two-stage preconcentration system, a low-volume capillary reactor and water trap, and a balanced reference gas delivery system. Measurements were performed on samples collected from two distinct sources (i.e. biogenic and vehicle emissions) and ambient air collected from downtown Miami and Everglades National Park. However, the instrumentation and the method have the capability to analyze a variety of source and ambient samples. The measured isotopic signatures that were obtained from source and ambient samples provide a new isotopic constraint for atmospheric chemists and can serve as a new way to evaluate their models and budgets for many OVOCs. In almost all cases, OVOCs emitted from fuel combustion were enriched in 13C when compared to the natural emissions of plants. This was particularly true for ethanol gas emitted in vehicle exhaust, which was observed to have a uniquely enriched isotopic signature that was attributed to ethanol’s corn origin and use as an alternative fuel or fuel additive. Results from this effort show that ethanol’s unique isotopic signature can be incorporated into air chemistry models for fingerprinting and source apportionment purposes and can be used as a stable isotopic tracer for biofuel inputs to the atmosphere on local to regional scales.
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Hoeven, Robin. "Investigating the contribution of protein dynamics to catalysis in protochlorophyllide oxidoreductase." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-contribution-of-protein-dynamics-to-catalysis-in-protochlorophyllide-oxidoreductase(029dda21-023f-4fdb-a980-26db7eab4833).html.
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Enzyme dynamics has been established to play a crucial role in catalysis, and it has therefore become an important area of research to better understand enzymatic rate enhancements. The light-activated enzyme protochlorophyllide oxidoreductase (POR) is a well-studied model system where dynamics are known to be important for catalysis. The catalytic reaction involves a sequential hydride and proton transfer to reduce the C17-C18 double bond in the protochlorophyllide (Pchlide) substrate with NADPH as a cofactor to yield the chlorophyllide (Chlide) product. Both H-transfer steps are established to undergo quantum tunneling, as derived from the temperature-dependence of the kinetic isotope effects (KIEs). Furthermore, a role for ‘promoting motions/vibrations’ has been presumed from the temperature-dependence KIE data, which will be investigated further in this thesis by the study of the KIE response to pressure. A general overview of the pressure-dependence as a new experimental probe is presented and compared with temperature-dependencies of KIEs, to establish whether pressure is suitable as an alternative technique for studying the role of enzyme dynamics in catalysis. This involves a comparison of pressure data from other enzyme systems to newly collected data for POR. However, no clear trend between temperature and pressure data is observed and hence, it can be concluded that pressure effects can be difficult to interpret. A case by case analysis is required and needs to be combined with computational simulations based on structural evidence (e.g. X-ray crystallographic), which is not yet available for POR.Solvent-viscosity has been successfully used to probe enzyme dynamics in POR and provides information on the extent of any protein networks that are involved along the reaction coordinate. Here I investigate the solvent-viscosity dependence of both H-transfer reactions in POR for a range of homologous POR enzymes to obtain an evolutionary perspective of the protein dynamics required for catalysis. This has been successfully used in the past on a limited number of POR homologues and has led to the formulation of a hypothesis supporting a twin-track evolution of the two catalytic steps in POR. I observed a lack of solvent-viscosity dependence in case of the hydride transfer across all the investigated lineages, while the proton transfer was shown to be more strongly affected by viscosity in prokaryotic enzymes than in their eukaryotic counterparts. This supports the proposed theory, suggesting an early optimisation of the dynamics involved in the light-activated hydride transfer with a strong reliance on localised motion. Conversely, the proton transfer experienced selective pressure to reduce its dependence on complex solvent-slaved motion and that has led to localised dynamics in eukaryotic POR homologues. Additionally, I found that the enzymes from eukaryotic species have a higher rate of both H-transfer steps, suggesting that an optimisation of the active site architecture occurred upon endosymbiosis. Enzyme dynamics clearly have a pivotal role to play in catalysis of this unique light-activated enzyme and detection of these will only be possible by detailed structural information.
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Wang, Zhen. "Catalytic mechanisms of thymidylate synthases: bringing experiments and computations together." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/2654.
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The relationship between protein structure, motions, and catalytic activity is an evolving perspective in enzymology. An interactive approach, where experimental and theoretical studies examine the same catalytic mechanism, is instrumental in addressing this issue. We combine various techniques, including steady state and pre-steady state kinetics, temperature dependence of kinetic isotope effects (KIEs), site-directed mutagenesis, X-ray crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations, to study the catalytic mechanisms of thymidylate synthase (TSase). Since TSase catalyzes the last step of the sole intracellular de novo synthesis of thymidylate (i.e. the DNA base T), it is a common target for antibiotic and anticancer drugs. The proposed catalytic mechanism for TSase comprises a series of bond cleavages and formations including activation of two C-H bonds: a rate-limiting C-H→C hydride transfer and a faster C-H→O proton transfer. This provides an excellent model system to examine the structural and dynamic effects of the enzyme on different C-H cleavage steps in the same catalyzed reaction. Our experiments found that the KIE on the hydride transfer is temperature independent while the KIE on the proton transfer is temperature dependent, implying the protein environment is better organized for H-tunneling in the former. Our QM/MM calculations revealed that the hydride transfer has a transition state (TS) that is invariable with temperature while the proton transfer has multiple subsets of TS structures, which corroborates with our experimental results. The calculations also suggest that collective protein motions rearrange the network of H-bonds to accompany structural changes in the ligands during and between chemical transformations. These computational results not only illustrate functionalities of specific protein residues that reconcile many previous experimental observations, but also provide guidance for future experiments to verify the proposed mechanisms. In addition, we conducted experiments to examine the importance of long-range interactions in TSase-catalyzed reaction, using both kinetic and structural analysis. Those experiments found that a remote mutation affects the hydride transfer by disrupting concerted protein motions, and Mg2+ binds to the surface of TSase and affects the hydride transfer at the interior active site. Both our experiments and computations have exposed interesting features of ecTSase that can potentially provide new targets for antibiotic drugs targeting DNA biosynthesis. The relationship between protein structure, motions, and catalytic activity learned from this project may have general implications to the question of how enzymes work.
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Huang, Min. "Elucidation des Mécanismes de O- et C-glycosylation par des Moyens Chimiques et Spectroscopiques." Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00923152.
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L'effet isotopique cinétique (KIE) est un outil puissant pour obtenir un aperçu sur le mécanisme d'une grande variété réactions. Nous avons observé différentes mesures de l'effet isotopique cinétique primaire du 13C pour la formation des α-, et β-mannopyranosides et des α- et β-glucopyranosides, en partant du sulfoxyde de glycosyle protégé par le groupement 4,6-O-benzylidène, par la spectroscopie RMN à ultrahaut champ (13C à 200 MHz et 1H à 800 MHz). Nous avons aussi calculé les KIE pour ces réactions en collaboration avec le Prof. Pratt à l'Université d'Ottawa. Les valeurs expérimentale et calculée (B3LYP / 6-31G (d, p) avec un modèle de continuum polarisable) sont en bon accord sauf pour l'α-mannopyranoside. Trois cas (-mannopyanoside, et -glucopyranosides) parmi les quatre ont montré un caractère "SN2-like", mais la formation de l'-mannopyranoside suggère fortement un mécanisme dissociatif (SN1). Une telle différence de mécanisme nécessite une authentification par des mesures cinétiques. Nous avons ensuite porté notre attention sur le développement d'une réaction intramoléculaire, comme horloge intramoléculaire, afin d'évaluer la cinétique relative des réactions de glycosylation. La formation des produits tricycliques fournit une grande évidence de l'existence d'un ion mannosyloxocarbénium comme un intermédiaire transitoire. Les réactions de compétition avec de l'isopropanol et du méthallyltriméthylsilane sont interprétées comme indiquant que la β-O-mannosylation passe par un mécanisme associatif (SN2-like), tandis que l'α-O-mannosylation et le β-C-mannosylation sont dissociative (SN1-like). Ceci est en plein accord avec nos résultats expérimentaux sur l'effet isotopique cinétique. Cette approche de la détermination de la cinétique relative des réactions de glycosylation est une méthode directe et est potentiellement applicable à une large variété de donneurs de glycosyle.
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Piansawan, Tammarat [Verfasser]. "Temperature Dependence of Carbon Kinetic Isotope Effect for the Oxidation Reaction of Ethane by Hydroxyl Radicals Under Atmospherically Relevant Conditions: Experimental and Theoretical Studies / Tammarat Piansawan." Wuppertal : Universitätsbibliothek Wuppertal, 2016. http://d-nb.info/1120337615/34.
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Ryberg, Per. "Concerted or Stepwise? : β-Elimination, Nucleophilic Substitution, Copper Catalysed Aziridination and Ruthenium Catalysed Transfer Hydrogenation Studied by Kinetic Isotope Effects and Linear Free-Energy Relationships." Doctoral thesis, Uppsala universitet, Avdelningen för organisk kemi, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2008.
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This thesis describes the use of kinetic isotope effects, linear free energy relationships and stereochamical studies to distinguish between different mechanistic alternatives and to obtain information about transition state structure. In the first part fluorine and deuterium kinetic isotope effects were determined for the base promoted HF elimination from 4-fluoro-4-(4’-nitrophenyl)butane-2-on. During this work a new method for the determination of fluorine kinetic isotope effects was developed. The results from the study demonstrates that the reaction proceeds via an E1cBip mechanism. In the second part the transition state structure for the SN2 reaction between ethyl chloride and cyanide ion in DMSO was studied. Kinetic isotope effects for six different positions in the reacting system, both in cyanide and ethyl chloride, were determined. The experimental isotope effects were then compared with the theoretically predicted isotope effects. The third part describes the use of Hammett type free-energy relationships and stereochemical evidence to study the mechanism of the copper catalysed alkene aziridination. The results from the study support a model that involves the simultaneous presence of two different copper nitrene intermediates. One which reacts non-stereospecifically via a radical intermediate and one which reacts stereospecifically via a concerted mechanism. In the fourth part a mechanistic study of the Ru(aminoalcohol) catalysed transfer hydrogenation of acetophenone in isopropanol is described. Kinetic isotope effects were determined for both proton and hydride transfer. The observation of significant primary deuterium kinetic isotope effects for both proton and hydride transfer support a mechanism where the proton and hydride are transferred simultaneously in a concerted mechanism.
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Wang, Zhihong. "Kinetic isotope effects, dynamic effects, and mechanistic studies of organic reactions." Texas A&M University, 2005. http://hdl.handle.net/1969.1/4886.
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Several organic reactions that could potentially involve coarctate transition states
were investigated by a combination of experimental and theoretical studies.
In the thermal fragmentation of âÂÂ-1,3,4-oxadiazolines, the mechanism supported by
kinetic isotope effects and theoretical calculations is a three-step process that does not
demonstrate any special stabilization in coarctate transition states. Rather than
undergoing a direct coarctate conversion to product, the mechanism avoids coarctate
steps. The last step is a concerted coarctate reaction, but being concerted may be viewed
as being enforced by the necessity to avoid high-energy intermediates.
In the deoxygenation of epoxides with dichlorocarbene, the stabilization from the
transition state aromaticity is not great enough to compete with the preference for
asynchronous bonding changes. KIEs and calculations suggested that the reaction occurs
in a concerted manner but with a highly asynchronous early transition state with much
more Cñ-O bond breaking than Cò-O bond breaking. In the Shi epoxidation, a large ò-olefinic 13C isotope effect and small ñ-carbon
isotope effect indicated an asynchronous transition state with more advanced formation
of the C-O bond to the ò-olefinic carbon. The calculated lowest-energy transition
structures are generally those in which the differential formation of the incipient C-O
bonds, the "asynchronicity," resembles that of an unhindered model, and the imposition
of greater or less asynchronicity leads to higher barriers. In reactions of cis-disubstituted
and terminal alkenes using Shi's oxazolidinone catalyst, the asynchronicity of the
epoxidation transition state leads to increased steric interaction with the oxazolidinone
when a ÃÂ-conjugating substituent is distal to the oxazolidinone but decreased steric
interaction when the ÃÂ-conjugating substituent is proximal to the oxazolidinone.
Dynamic effects were studied in Diels-Alder reaction between acrolein and methyl
vinyl ketone. This reaction yields two products in a ratio of 3.0 ñ 0.5. Theoretical studies
shows that only one transition structure is involved in the formation of both.
Quasiclassical trajectory calculations on an MP2 surface give a prediction of a product
ratio of 45:14 (3.2:1), which is in good agreement with the experimental observation.
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Guo, Weifu Blake Geoffrey A. Eiler John. "Carbonate clumped isotope thermometry : application to carbonaceous chondrites and effects of kinetic isotope fractionation /." Diss., Pasadena, Calif. : California Institute of Technology, 2009. http://resolver.caltech.edu/CaltechETD:etd-12182008-115035.
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Li, Jiayue. "The preservation of protein dynamics from bacteria to human dihydrofolate reductase." Diss., University of Iowa, 2019. https://ir.uiowa.edu/etd/6984.
Full textAbstract:
Protein motions are complex, including occurring at different time scales, and their roles in enzyme-catalyzed reactions have always been of great interest among enzymologists. In order to characterize the potential factors that play a role on the chemical step of enzymatic reactions, variants of dihydrofolate reductase have been used as a benchmark system to study the motions of proteins correlated with the chemical step. A “global dynamic network” of coupled residues in Escherichia coli dihydrofolate reductase (ecDHFR), which assists in catalyzing the chemical step, has been demonstrated through quantum mechanical/molecular mechanical and molecular dynamic (QM/MM/MD) simulations, as well as bioinformatic analyses. A few specific residues — M42, G121, and I14 — were shown to function synergistically with measurements of single turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. Although similar networks have been found in other enzymes, the general features of these networks are still unclear. This project focuses on exploring homologous residues of the proposed global network in human DHFR through computer simulations and measurements of the temperature dependence of KIEsint. The mutants M53W and S145V, both remote residues, showed significant decreases in catalytic efficiency. Non-additive isotope effects on activation energy were observed between M53 and S145, indicating their synergistic effect on hydride transfer in human DHFR.
Apart from the effects of the conserved residues, we also extend our studies to exploring three potential phylogenetic events that account for the discrepancies between E. coli and human DHFR. They are L28, PP insertion and PEKN insertions by phylogenetic sequence analysis. Two of them (N23PP and G51PEKN E. coli DHFR) have been proved to be important both by MD simulation and experimental probe of KIEs measurement. The experiments have found that PP insertion itself rigidified the M20 loop and motions coupled to hydride transfer were impaired, however, loop rigidification was improved after incorporating PEKN. Furthermore, deletion of PP and PEKN of the engineered human enzyme also show a similar outcome. However, the effect of the key residue of L28 is not clear. In this project, we have step-wise engineered the human DHFR to be like hagfish (F31M) and E. coli (F32L). And it is found out that there is an increase in the temperature dependence of KIEs when the enzyme was bacterilized into a more primitive variant. This indicates that not only is residue F32 important and correlated with the chemical step as indicated by bioinformatic studies, but it is possible to trace the evolutionary trajectory. A triple mutation F32L-PP26N-PEKN62G on the human DHFR was also conducted, and it is not surprising to find out that the temperature dependence of KIEs has retained its behavior like wild-type human DHFR. These results suggest that the three predicted phylogenetically coherent events coevolved together to maintain the evolutionary preservation of the protein dynamics to enable H-tunneling from well-reorganized active sites.
As has been indicated by the previous project, as the enzyme evolves, the active site of the enzyme will “reorganize” to form the optimal transition state for chemical step (from F32L-F32M-wild type DHFR). Here in this project, we aimed to systematically address this point of view through a series of cyclic permutation DHFR from directed evolutions. As this primitive enzyme is 7 orders of magnitude less efficient than the well-evolved human DHFR, together with four generations of evolved variants (cp, cp’ and cp”), this provides a good model system for explorations of the molecular basis of enzyme evolution. It is found that the organizations of transition state are improved before the catalytic efficiency is enhanced as the enzyme evolves.
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Jenson, David L. Jenson. "Proton-coupled electron transfer and tyrosine D of phototsystem II." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/29667.
Full textAbstract:
Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2010.Committee Chair: Bridgette Barry; Committee Member: Ingeborg Schmidt-Krey; Committee Member: Jake Soper; Committee Member: Nils Kroger; Committee Member: Wendy Kelly. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Backstrom, Nicholas. "Primary Kinetic Isotope Effects in Intramolecular Deprotonation of Carbon Acids." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511907.
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Hothi, Parvinder. "Multiple ligand binding and kinetic isotope effects in methanol dehydrogenase." Thesis, University of Leicester, 2004. http://hdl.handle.net/2381/29691.
Full textAbstract:
The reaction of PQQ-dependent methanol dehydrogenase (MDH) has been studies by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and kinetic isotope effects (KIEs). Phenazine ethosulphate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site. MDH activity is dependent on activation by ammonium, bit is inhibited at high ammonium concentrations. Glycine ethyl ester (GEE; an alternative activator) does not inhibit enzyme activity to the same degree as ammonium. Data suggest a close spatial relationship between the Ks and Ki activator-binding sites such that binding of GEE to the Ks site sterically impairs binding of GEE to the Ki site. Cyanide is less effective at suppressing endogenous activity with GEE, which is attributed to impaired binding of cyanide at the catalytic site through steric interaction with GEE at the Ks site. Combined, this data suggests that the catalytic methanol-binding site and the K s and Ki sites are in close proximity. PES and substrate influence the stimulatory and inhibitory effects of activator through competitive binding, giving rise to unusual KIEs as a function of activator and PES concentration. The KIEs are independent of temperature, consistent with the proposed competitive binding model. Stopped-flow studies indicate that at high ammonium concentrations, the oxidative half-reaction becomes more rate-limiting. With GEE, the oxidative half-reaction makes a larger contribution to rate limitation, stopped-flow method has enabled study of the reductive half-reaction of MDH. A kinetic mechanism for the reaction cycle of MDH is proposed. Furthermore, this study emphasises the need for caution in using KIEs, and the temperature dependence of KIEs, as a probe for enzymatic hydrogen tunnelling.
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Christian, Chad F. "The experimental and theoretical determination of combinatorial kinetic isotope effects for mechanistic analysis." [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1253.
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Ussing, Bryson Richard. "Systematic examination of dynamically driven organic reactions via kinetic isotope effects." Texas A&M University, 2006. http://hdl.handle.net/1969.1/4877.
Full textAbstract:
Organic reactions are systematically examined experimentally and theoretically to
determine the role dynamics plays in the outcome of the reaction. It is shown that
trajectory studies are of vital importance in understanding reactions influenced by
dynamical motion. This dissertation discusses how a combination of kinetic isotope
effects, theoretical calculations, and quasiclassical dynamics trajectories aid in the
understanding of the solvolysis of p-tolyldiazonium cation in water, the cycloadditions
of cyclopentadiene with diphenylketene and dichloroketene, and the cycloaddition of 2-
methyl-2-butene with dichloroketene.
In the solvolysis of p-tolyldiazonium cation, significant 13C kinetic isotope effects
are qualitatively consistent with a transition state leading to formation of an aryl cation,
but on a quantitative basis, the isotope effects are not adequately accounted for by simple
SN1 heterolysis to the aryl cation. The best predictions of the 13C isotope effects for the
heterolytic process arise from transition structures solvated by clusters of water
molecules. Dynamic trajectories starting from these transition structures afford products very slowly. The nucleophilic displacement process for aryldiazonium ions in water is
determined to be at the boundary of the SN2Ar and SN1 mechanisms.
The reaction of cyclopentadiene with diphenylketene affords both [4 + 2] and [2 +
2] cycloadducts directly. This is surprising. There is only one low-energy transition
structure for adduct formation. Investigation of this reaction indicates that quasiclassical
trajectories started from a single transition structure afford both [4 + 2] and [2 + 2]
products. Overall, an understanding of the products, rates, selectivities, isotope effects,
and mechanism in these reactions requires the explicit consideration of dynamic
trajectories.
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Roston, Daniel Harris. "The use of kinetic isotope effects in studies of hydrogen transfers." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/1498.
Full textAbstract:
The present dissertation seeks to deepen our understanding of hydrogen transfers and especially C-H bond activations in enzymes. Hydrogen transfers are ubiquitous in chemistry and biology and a thorough understanding of how they occur and what factors influence them will facilitate developments in biomimetic catalysis, rational drug design, and other fields. A particular difficulty with H-transfers is the importance of nuclear quantum effects to the reaction, particularly tunneling. The overall scope of the work here aims to examine how experimental kinetic isotope effects (KIEs) can be interpreted with a particular type of tunneling model, referred to as Marcus-like models, to yield a semi-quantitative picture of the physical mechanisms of H-transfers. Previous work had used this kind of model to qualitatively interpret experimental data using a combination of intuition and generalized theories. The work here examines these theories in quantitative detail, testing and calibrating our intuition in the context of several experimental systems. The first chapter of research (ch. II) focusses on the temperature dependence of primary KIEs and how these experiments can be quantitatively interpreted as a probe for certain kinds of enzyme or solvent dynamics. The subsequent chapters (ch. III-VI) focus on the use of secondary KIEs to determine the detailed structures of tunneling ready states (TRSs) and how the dynamics of H-tunneling affect those structures. These chapters focus primarily on the TRS of the enzyme alcohol dehydrogenase, but by examining an uncatalyzed analogue to that reaction (ch. VI), the work gains some insight about similarities and differences between catalyzed and uncatalyzed reactions. In summary, the work uncovers some principles of catalysis, not just the mechanism of a catalyzed reaction. The mechanism of C-H activation presented here provides an elegant solution to problems that have been vexing to accommodate within traditional models. This work constitutes some initial steps in making Marcus-like models quantitatively useful as a supplement or even replacement for traditional models of reactivity.
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Sobrado, Pablo. "Studies of the chemical mechanisms of flavoenzymes." Texas A&M University, 2003. http://hdl.handle.net/1969/565.
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Owens, Simon. "Kinetics and mechanisms of hydrogen isotope exchange over solid storage media." Thesis, University of Bath, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.687343.
Full textAbstract:
Hydrogen isotope separation systems using palladium (Pd) are currently being designed for both reactor designs with the aim of separating and purifying the reactor exhaust products which contain valuable unspent hydrogen isotopes. Hydrogen isotope exchange in Pd offers an efficient, ambient condition process that can produce pure isotopic species in a process far simpler and less costly than the current state of the art cryogenic distillation processes. The method is applicable whether separating hydrogen (protium), deuterium or tritium and any combination of these. If practical fusion devices are ever to be realised it is essential to produce an economical and efficient fuel cycle capable of separating and purifying hydrogen isotopes. Hydrogen isotope exchange in Pd is also of interest to the waste separation and purification industries, in particular those using hydrogen separation membranes which used Pd and Pd-alloy membranes. Understanding hydrogen isotope exchange, with particular regard to the formation of the intermediate (and often unwanted) hydrogen deuteride (HD), will aid significantly in future designs of hydrogen isotope separation systems. Novel hydrogen isotope exchange experiments involving hydrogen and deuterium at a number of temperatures (208 K, 293 K and 373 K) and pressures (1.3 bar – 8 bar) not yet explored are presented in this thesis. The experiments were carried out on a unique piece of laboratory apparatus provided to and further developed at the University of Bath. Alongside experimentation, a novel comprehensive multidimensional multi-physics model has been created to analyse the experimental data obtained using the new apparatus and elucidate the kinetics and mechanisms of the reactions occurring between hydrogen isotopic species and Pd during hydrogen isotope exchange based on Langmuir-Hinshelwood surface reaction mechanism. The surface reaction rates, kinetic rate constants and heat effects have been examined in detail, and in tandem, for the first time.
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Frantom, Patrick Allen. "Studies of the chemical and regulatory mechanisms of tyrosine hydroxylase." Texas A&M University, 2006. http://hdl.handle.net/1969.1/3817.
Full textAbstract:
Tyrosine hydroxylase (TyrH) catalyzes the pterin-dependent hydroxylation of
tyrosine to form dihydroxyphenylalanine. The enzyme requires one atom of ferrous iron
for activity. Using deuterated 4-methylphenylalanine substrates, intrinsic primary and
secondary isotope effects of 9.6 ± 0.9 and 1.21 ± 0.08 have been determined for benzylic
hydroxylation catalyzed by TyrH. The large, normal secondary isotope effect is
consistent with a mechanism involving hydrogen atom abstraction to generate a radical
intermediate. The similarity of the isotope effects to those measured for benzylic
hydroxylation catalyzed by cytochrome P-450 suggests that a high-valent, ferryl-oxo
species is the hydroxylating species in TyrH. Uncoupled mutant forms of TyrH have
been utilized to unmask isotope effects on steps in the aromatic hydroxylation pathway
which also implicate a ferryl-oxo intermediate. Inverse secondary isotope effects were
seen when 3,5-2H2-tyrosine was used as a substrate for several mutant enzyme forms.
This result is consistent with a direct attack by a ferryl-oxo species on the aromatic ring
of tyrosine forming a cationic intermediate. Rapid-freeze quench Mössbauer studies have provided preliminary spectroscopic evidence for an Fe(IV) intermediate in the reaction
catalyzed by TyrH.
The role of the iron atom in the regulatory mechanism has also been investigated.
The iron atom in TyrH, as isolated, is in the ferric form and must be reduced for activity.
The iron can be reduced by a number of one-electron reductants including
tetrahydrobiopterin, ascorbate, and glutathione; however, it appears that BH4 (kred = 2.8 ±
0.1 mM-1 s-1) is the most likely candidate for reducing the enzyme in vivo. A one-electron
transfer would require a pterin radical. Rapid-freeze quench EPR experiments aimed at
detecting the intermediate were unsuccessful, suggesting that it decays very rapidly by
reducing another equivalent of enzyme. The active Fe(II) form can also become oxidized
by oxygen (210 ± 30 M-1 s-1); this increases the affinity of catecholamine inhibitors.
Serine 40 can be phosphorylated to relieve the inhibition; however, results with S40E
TyrH show phosphorylation does not have an effect on the rate constant for reduction of
the enzyme but causes a 40% decrease in the rate constant of oxidation.
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Kellerman, Daniel. "Application of Internal Competition Kinetics to Probe the Catalytic Strategies of RNA 2’-O-transphosphorylation." Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1449150064.
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Ma, Li. "Study of the Primary Isotope Dependence of Secondary Kinetic Isotope Effects in Hydride Transfer Reactions in Solution." Thesis, Southern Illinois University at Edwardsville, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10843728.
Full textAbstract:
It has been accepted that hydrogen-transfer reactions take place through a quantum mechanical tunneling mechanism, where H tunnels through its classical energy barrier in light of its wave form. There are several H-tunneling models proposed, including the contemporary Marcus-like H-tunneling model, which explains that the donor-acceptor distance (DAD) in the tunneling ready state (TRS) is shorter for a heavier isotope (e.g. deuterium (D)) then a lighter isotope (e.g., protium (H)). This model has been used to explain the kinetic isotopic effect observations in H-tunneling processes to provide mechanistic role of protein in enzyme catalysis. The purpose of the research is to test the hypothesis of “isotopically different DAD” concept by studying the hydrogen/deuterium-transfer reactions in solutions, given that hydride-transfer reactions account for over 50% of biological reactions. Our group’s previous results showed that the steric hindrance and hydrogen-bonding effect played a significant role in the different hydrogen vs deuterium tunneling-ready states. In general, the shorter DAD creates more spatial crowding effect which will affect the 2° C-H vibrations and decrease the 2° KIEs. In this thesis, different reaction systems were designed to test these effects by studying the 1° isotope dependence of 2° KIEs at the near and remote positions from the reaction center. It was found that the results are consistent with the hypothesis of the “isotopically different TRS structures”.
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Pudney, Christopher R. "Kinetic isotope effects as probes of the mechanism of enzymatic hydrogen transfer." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492045.
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Nowlan, Daniel Thomas. "Mechanisms of transition-metal catalyzed additions to olefins." Texas A&M University, 2003. http://hdl.handle.net/1969.1/2308.
Full textAbstract:
Transition metal catalyzed reactions have an important place in synthetic chemistry, but the
mechanistic details for many of these reactions remain undetermined. Through a combination of
experimentally determined 13C kinetic isotope effects (KIEs) and density functional theory (DFT)
calculations, some of these reactions have been investigated.
The cyclopropanation of an olefin catalyzed by rhodium (II) tetrabridged complexes has been shown
to proceed through an asynchronous, but concerted mechanism. DFT does not provide an accurate
transition structure for the reaction of an unstabilized carbenoid with an olefin, but it does predict an
early, enthalpically barrierless transition state which is consistent with the reactivity of unstabilized
carbenoids. For the case of stabilized carbenoids, the theoretical structures predict the KIEs accurately
and a new model is proposed to explain the selectivity observed in Rh2(S-DOSP)4-catalyzed
cyclopropanations.
The chain-elongation step of atom transfer radical polymerization (ATRP) has been shown to be
indistinguishable from that of free radical polymerization (FRP) for the CuBr/2,2??-bipyridine system.
While DFT calculations predict an earlier transition state than observed, the calculations suggest that
with increasing levels of theory the predicted KIEs come closer to the observed KIEs.
A recently proposed [2 + 2] mechanism for the cyclopropenation of alkynes catalyzed by
Rh2(OAc)(DPTI)3 has been shown not to be a viable pathway. Rather, the experimental KIEs are
predicted well by canonical variational transition state theory employing the conventional mechanism for
cyclopropenation via a tetrabridged rhodium carbenoid. DFT calculations also suggest an alternative
explanation for the observed enantioselectivity.
The 13C KIEs for metal-catalyzed aziridination have been measured for three separate catalytic
systems. While the KIEs do not completely define the mechanism, all of the reactions exhibit similar
KIEs, implying similar mechanisms. A surprising feature of this system is the presumed nitrene
intermediate??s triplet spin state. This complicates the DFT analysis of this system.
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Leavitt, William Davie. "On the mechanisms of sulfur isotope fractionation during microbial sulfate reduction." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11511.
Full textAbstract:
Underlying all applications of sulfur isotope analyses is our understanding of isotope systematics. This dissertation tests some fundamental assumptions and assertions, drawn from equilibrium theory and a diverse body of empirical work on biochemical kinetics, as applied to the multiple sulfur isotope systematics of microbial sulfate reduction. I take a reductionist approach, both in the questions addressed and experimental approaches employed. This allows for a mechanistic, physically consistent interpretation of geological and biological sulfur isotope records. The goal of my work here is to allow interpreters a more biologically, chemically and physically parsimonious framework to decipher the signals coded in modern and ancient sulfur isotope records.Earth and Planetary Sciences
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Rungsrisuriyachai, Kunchala. "On the Catalytic Roles of HIS351, ASN510, and HIS466 in Choline Oxidase and the Kinetic Mechanism of Pyranose 2-Oxidase." Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/chemistry_diss/36.
Full textAbstract:
Choline oxidase (E.C. 1.1.3.17) from Arthrobacter globiformis catalyzes the four-electron oxidation of choline to glycine betaine (N,N,N-trimethylglycine) via two sequential, FAD-dependent reactions in which betaine aldehyde is formed as an enzyme-bound intermediate. In each oxidative half-reaction, molecular oxygen acts as electron acceptor and is converted into hydrogen peroxide. Biochemical, structural, and mechanistic studies on the wild-type and a number of mutant variants of choline oxidase have recently been carried out, allowing for the depiction of the mechanism of alcohol oxidation catalyzed by the enzyme. Catalysis by choline oxidase is initiated by the removal of the hydroxyl proton of alcohol substrate by a catalytic base in the enzyme-substrate complex, yielding the formation of the alkoxide species. In this dissertation, the roles of His351 and conserved His466 were investigated. The results presented demonstrate that His351 is involved in the stabilization of the transition state for the hydride transfer reaction and contributes to substrate binding. His466 is likely to be a catalytic base in choline oxidase due to its dramatic effect on enzymatic activity. Comparison of choline oxidase and other enzymes within its superfamily reveals the presence of a conserved His-Asn pair within the active site of enzymes. Therefore, the role of the conserved Asn510 in choline oxidase was examined in this study. The results presented here establish the importance of Asn510 in both the reductive and oxidative half-reactions. The lost of ability to form a hydrogen bond interaction between the side chain at position 510 with neighboring residues such as His466 resulted in a change from stepwise to concerted mechanism for the cleavages of OH and CH bonds of choline, as seen in the Asn510Ala mutant. Finally, the steady-state kinetic mechanism of pyranose 2-oxidase in the pH range from 5.5 to 8.5 was investigated. It was found that pH exerts significant effects on enzyme mechanism. This study has established the involvement of the residues in the initiation of enzyme catalysis and the stabilization of the alkoxide intermediate in choline oxidase. In addition, this work demonstrates the first instance in which the kinetic mechanism of a flavin-dependent oxidase is governed by pH.
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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/.
Full textAbstract:
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.
36
Guo, Qi. "Enzyme dynamics and their role in formate dehydrogenase." Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/2216.
Full textAbstract:
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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Lavigne, André. "Oxydations cupro-catalysees des amines aliphatiques : etudes mecanistiques et applications synthetiques." Paris 6, 1987. http://www.theses.fr/1987PA066470.
Full textAbstract:
Preparation de nitriles a partir d'amines primaires et d'alpha -aminoacides. Les acides amines monosubstutitues rch(nh::(2))co::(2)h donnent le nitrile rcn, alors que les acides amines disubstitues rr'c(nh::(2))co::(2)h conduisent a l'azine rr'c=n-n=cr'r. Les mecanismes proposes font intervenir le cuivre (iii), forme in situ a partir du cuivre (i) et de l'oxygene
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Ralph, Erik C. "The chemical mechanisms of flavin-dependent amine oxidases and the plasticity of the two-his one-carboxylate facial triad in tyrosine hydroxylase." [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1043.
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Uluisik, Rizvan C. "Effects of Temperature on the Kinetic Isotope Effects for Proton and Hydride Transfers in the Active Site Variant of Choline Oxidase Ser101Ala." Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/chemistry_theses/56.
Full textAbstract:
Choline oxidase catalyzes the oxidation of choline to glycine betaine. The reaction includes betaine aldehyde as an intermediate. FAD is reduced by the alcohol substrate, betaine aldehyde intermediate and oxidized by molecular oxygen to give hydrogen peroxide. In this study, the Ser101Ala variant of choline oxidase was prepared to elucidate the contribution of the hydroxyl group of Ser101 in the proton and hydride transfer reactions for proper preorganization and reorganization of the active site towards quantum mechanical tunneling. The thermodynamic parameters associated with the enzyme-catalyzed OH and CH bond cleavages and the temperature dependence of the associated solvent and substrate kinetic isotope effects were investigated using a stopped-flow spectrophotometer. The proton and hydride transfer have been shown to be occurring via quantum tunneling in CHO-S101A enzyme.
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Gurevic, Ilya. "Studies on the hydride transfer and other aspects of several thymidylate synthase variants." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6586.
Full textAbstract:
The nucleotide 2'-deoxythymidine 5'-monophosphate (thymidylate, dTMP) is phosphorylated twice to become a substrate for DNA polymerases, which copy a cell’s genetic information in advance of cell division. The main route to dTMP is mediated by the enzyme thymidylate synthase (TSase) and goes through 2'-deoxyuridine 5'-monophosphate (dUMP); dUMP’s heterocyclic aromatic pyrimidine ring loses a proton from its C5 position and gains a methylene and a hydride from the other reactant, methylene tetrahydrofolate (MTHF). In general, intricate knowledge of an enzyme’s mechanism can yield insight that leads to the development of precision-targeted inhibitors tailored exactly to thymidylate synthase. In fact, even more careful targeting could be achievable: Although E. coli TSase has served as a model system, investigators have increasingly been directing their lines of inquiry toward human TSase.
A general enzymatic catalytic cascade is complex, comprising substrate binding, the chemical steps and product release; typically, the product release step is rate-limiting. TSase, however, is partially rate-limited by the chemistry portion of the process. The enzymatic mechanism has been considered for decades, yet recently has undergone a reassessment. After substrate binding – for which there is strong evidence for preference to dUMP as the first ligand in the wild-type E. coli enzyme – the important events are methylene transfer from MTHF to dUMP, proton abstraction and hydride transfer. The last of these – hydride transfer – is irreversible and rate-limiting (to a large degree without Mg2+, and to a small but noticeable degree with Mg2+). The studies described here are aimed at three therapeutically relevant questions: (a) determining the extent of negative charge accumulation at the O4 position of the hydride transfer acceptor; (b) expanding knowledge of the differential properties of E. coli and human TSase; and (c) gaining insight into the molecular origin of the drug resistance seen in a clinically relevant human TSase mutant.
The properties touched on in this work include steady-state kinetics; inhibition constants toward 5-fluoro dUMP, substrate binding sequence and the temperature dependency of intrinsic hydride transfer kinetic isotope effects (KIEs). Intrinsic KIEs are a specialized measurement that permits the investigator to examine a particular hydrogen transfer step in isolation; it is achieved by labeling the bond to hydrogen broken in the reaction with protium (1H, also written as H), deuterium (2H, also written as D) or tritium (3H, also written as T). The latter is radioactive. The reaction is conducted with a mixture of two hydrogen isotopes at a time, and the extent to which the heavier isotope is disfavored against reaction is assessed; this covers multiple steps. Heavier isotopes directly participating in a chemical step react slower both because of zero-point vibrational energies if a semi-classical view is taken and because of the mass-dependence of tunneling probabilities if a quantum-mechanical view is taken. Each of the two-way isotopic comparisons mentioned above furnishes an observed KIE for that competition between two isotopes. Mathematical combination of two isotopic comparisons cancels out the effect of isotopically insensitive steps and provides rich insight into the hydride transfer alone. The ultimate result is the ratio of rate constants for the isotopologues; this ratio’s magnitude and variation with temperature report on the compactness of the active site and its resistance to thermal fluctuation, respectively.
Our results reveal a possible role for E. coli asparagine 177 (N177) in the hydride transfer transition state (TS) stabilization, as revealed by its disruption in the aspartate mutant, N177D. This disruption was found to be alleviated to a high extent when the substrate was changed to dCMP, consistent with the N177 stabilizing partial negative charge at the TS for hydride transfer. This has drug design implications. Our work on human TSase underscores slightly weaker substrate binding preference, insensitivity to Mg2+ and mild alteration of hydride transfer TS when compared with E. coli TSase. Finally, analysis of the Y33H mutant of human TSase – the affected residue being remote from the active site – indicated the drug resistance was because of a higher inhibition constant for 5F-dUMP and that the hydride transfer step is disrupted, with a wider variation among donor-acceptor distances (between the two carbons involved in the hydride transfer at the TS for that step). Other researchers’ crystallographic evidence reveals greater positional uncertainty for a set of active-site side chains in the E. coli equivalent mutant. In totality, the data available implicate enzyme motions as relevant to drug binding and to catalysis for human TSase.
In summary, the research described herein enriches the understanding of several aspects of the behavior of multiple TSase variants – the overall performance as seen via steady-state kinetics; the pattern of substrate binding as seen with observed KIEs for the proton abstraction step; and the efficiency of active site preparation for hydride transfer as evidenced in the temperature dependency of intrinsic hydride transfer KIEs.
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Roberts, Kenneth M. "Mechanistic evaluation of N-dealkylation by cytochrome P450 using N,N-dimethylaniline N-oxides and kinetic isotope effects." Pullman, Wash. : Washington State University, 2009. http://www.dissertations.wsu.edu/Dissertations/Fall2009/k_roberts_113009.pdf.
Full text
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Mauve, Caroline. "Production et hydrolyse des amides : mécanismes chimiques, isotopie et applications : étude de la glutamine synthétase." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112397.
Full textAbstract:
La nutrition azotée des bactéries et des plantes est actuellement un sujet de grande importance, notamment pour comprendre comment améliorer les voies métaboliques aboutissant à l’assimilation de l’azote et à plus grande échelle, optimiser des apports d’engrais et augmenter le rendement des cultures. Dans ce contexte, la réaction d’amidation catalysée par la glutamine synthétase (GS), qui fixe l’ammonium (NH₄)⁺ en glutamine, est cruciale car elle est à la fois le point d’entrée de l’azote dans les végétaux, et une étape-clef du recyclage de l’azote (en particulier, NH₄⁺ photorespiratoire). Dans cette étude, nous nous sommes intéressés à la cinétique enzymatique et au mécanisme chimique de la GS. Des systèmes analytiques (HPLC, RMN , GC-MS) ont été optimisés pour permettre la mesure de l’activité enzymatique in vitro et pour réaliser des analyses par spectrométrie de masse à ratio isotopique. Avec ces techniques, nous avons pu regarder précisément les effets isotopiques ¹²C/¹³C, ¹⁴N/¹⁵N et H₂O/D₂O (solvant) lors de la catalyse, en utilisant la GS d’E. coli et d’Arabidopsis thaliana (GS1,2). Nos résultats montrent qu’il n’y a pas d’effet isotopique ¹²C/¹³C, mais qu’il y a un fractionnement ¹⁴N/¹⁵N de »16‰. En outre, il y a un effet inverse du solvant (réaction 1.5 à 2 fois plus rapide dans D₂O). Cela suggère que la création de la liaison C----N (amidation) est partiellement limitante (engagement catalytique de »14% seulement) et que le réseau de ponts hydrogènes dans le site actif est crucial pour déterminer la vitesse de la réaction. L’apparition d’effets ¹⁴N/¹⁵N inverses dans certaines circonstances et les effets drastiques causés par une substitution du cofacteur métallique (Mg²⁺) suggèrent en outre que l’étape d’amidation peut être réversible et que la coordination par un métal joue un rôle très important pour stabiliser les intermédiaires de la réaction, en interaction avec le solvant. Ainsi, dans son solvant naturel qu’est H₂O, la GS réalise une réaction ‘chimiquement difficile’ (barrière énergétique élevée de l’amidation) rendue possible par le clivage de l’ATP et son caractère exergoniqueNitrogen nutrition in bacteria and plants is currently an important topic, in particular to identify key points for metabolic improvements in N assimilation and more generally, to optimize fertilization and crop yield. In such a context, the amidation reaction catalyzed by glutamine synthetase (GS), which fixes ammonium (NH₄)⁺ into glutamine, is of crucial importance since it both represents the N entry in plants and the main step of N recycling (such as photorespiratory (NH₄)⁺. Here, we examined GS kinetics and chemical mechanism. Analytical methods (HPLC, NMR, GC-MS) have been set up so as to measure in vitro activities and isotopic abundance by isotope ratio mass spectrometry. These gave access to isotope effects (¹²C/¹³C, ¹⁴N/¹⁵N et H₂O/D₂O – solvent) during catalysis, with the GS from either E. coli or A. thaliana (GS1,2). Our results show that there no ¹²C/¹³C isotope effect but there is significant ¹⁴N/¹⁵N isotope fractionation of ca. 16‰. In addition, there is an inverse solvent isotope effect (reaction 1.5 to 2 times faster in D₂O). This suggests that forming the C----N bond (amidation) is partially rate-limiting (catalytic commitment of ca. 14% only) and the H-bond network in the active site is of substantial importance for the reaction rate. The occurrence of inverse ¹⁴N/¹⁵N isotope effects under certain circumstances as well as the drastic impact of changing the metal cofactor (Mg²⁺)) indicate that the amidation step can be reversible and that the coordination by the metal plays a key role in stabilizing reaction intermediates, by interfacing the solvent. In other words, in its natural solvent H₂O, the GS catalyses an intrinsically ‘difficult’ reaction (high energy barrier of amidation) made possible by both ATP cleavage and its exergonic nature
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Hatzakorzian, Roupen. "The effect of insulin on whole body protein and glucose metabolism after cardiac surgery using stable isotope kinetics: a pilot study." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107612.
Full textAbstract:
High doses of insulin are required to prevent the hyperglycemic response to open heart surgery. Insulin has been shown to produce hypoaminoacidemia during coronary artery bypass graft (CABG) surgery. The present study investigated the effect of high-dose insulin therapy on whole body protein and glucose metabolism in patients undergoing CABG surgery using stable isotope tracers [6,6-2H2]glucose and L-[1-13C]-leucine. Hyperinsulinemic-normoglycemic clamp was used to deliver high-dose insulin. The primary objective was to establish a protocol and to assess the feasibility of using stable isotope tracers after cardiac surgery while continuously infusing high-dose insulin. The effect of high-dose insulin on protein breakdown, synthesis and oxidation and glucose turnover was evaluated.Fifteen patients were recruited to the study, seven in the control group receiving standard glycemic control and eight in the insulin group receiving the hyperinsulinemic-normoglycemic clamp. Isotopic plateaus of L-[1-13C]leucine, [6,6-2H2]glucose and expired 13CO2 were attained in all patients in both groups with a coefficient of variance <5%. Protein breakdown and synthesis both decreased in patients who received high-dose insulin, while protein oxidation remained the same, resulting in a negative protein balance regardless of treatment group. Endogenous glucose production was almost completely suppressed by the administration of high-dose insulin and 20% dextrose solution. The hyperinsulinemic-normoglycemic clamp is a metabolic intervention that when used in the perioperative setting of cardiac surgery, has significant effects on whole body protein breakdown.Lors de chirurgies pour pontage aorto-coronarien, l'utilisation de hautes doses d'insuline est habituellement nécessaire pour prévenir l'augmentation de la glycémie en réponse au stress chirurgical. Il a été démontré que l'administration d'insuline induit une hypoaminoacidémie chez ces patients. La présente étude a pour but d'évaluer les effets de l'administration de hautes doses d'insuline sur le métabolisme des protéines et du glucose par l'usage d'isotopes stables pour ce type d'intervention chirurgicale. Les isotopes utilisés étaient le [6,6-2H2]glucose et la L-[1-13C]leucine. L'objectif principal était de vérifier s'il est possible de faire le suivi postopératoire de ces marqueurs isotopiques en présence de hautes doses d'insuline, et ce afin d'établir un protocole. Les effets de ces hautes doses d'insuline sur la lyse, la synthèse et l'oxydation protéique ainsi que les effets sur le métabolisme du glucose ont été évalués. Quinze sujets ont été recrutés, soit sept dans le groupe témoin recevant le traitement habituel de contrôle de glycémie, et les huit autres dans le groupe d'étude soumis à des hautes doses d'insuline. Les plateaux du [6,6-2H2]glucose, de la L-[1-13C]leucine et du 13CO2 ont été obtenus pour tous les candidats des deux groupes, avec un coefficient de variance < 5%. La lyse et la synthèse protéiques des patients recevant de hautes concentrations d'insuline ont toutes deux diminuées alors que l'oxydation, quant à elle, est restée la même. Un bilan de protéines négatif a été obtenu quelque soit le groupe de traitement. La production endogène de glucose a pratiquement été abolie par l'administration de grande quantité d'insuline et de dextrose 20%. Cette intervention métabolique, lorsqu'utilisée dans le contexte de chirurgies cardiaques, a des effets significatifs sur le métabolisme global des protéines et du glucose des patients.
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Uliaque, Cugat Katia. "Implementation of stable isotopes lipoprotein kinetic studies: effects on HDL metabolism of a Mediterranean type diet rich in MUFAs from virgin olive oil." Doctoral thesis, Universitat Rovira i Virgili, 2007. http://hdl.handle.net/10803/8658.
Full textAbstract:
The anti-atherogenic effects ascribed to a Mediterranean-type diet rich in monounsaturatedfatty acids (MUFAs) from virgin olive oil are due, partly, to an increase in, or maintenance of,
plasma concentrations of high density lipoprotein (HDL) cholesterol. However, the underlying
mechanisms that may explain these concentrations are not well characterised, to-date.
Apolipoprotein (apo) A-I (apoA-I) is the major HDL apo and its kinetic parameters, such as
production rate and catabolic rate, reflect the kinetics of the HDL particle. Our working
hypothesis is as follows: a Mediterranean-type diet rich in MUFAs from virgin olive oil,
compared to a STEP II diet, increases or preserves HDL cholesterol concentrations due to an
increase in apoA-I production and not to a decrease in apoA-I catabolism. Kinetic studies
using stable isotopes are, perhaps, the best approach in physiologically evaluating apo
production and catabolism in humans. This methodology has not as yet been implemented in
Spain. Objectives: to implement the necessary methodology to perform kinetic studies of apo
B-100 and, especially, of apoA-I and apoA-II in volunteers in vivo using stable isotopes to
label proteins in vivo. Further, we used this methodology to analyse the overall effects of a
Mediterranean-type diet rich in monounsaturated fatty acids from virgin olive oil compared
with a low-fat, STEP II diet, on HDL and low density lipoprotein (LDL) metabolism. Design:
we conducted a crossover, randomised study with dietary intervention periods of 4 weeks,
interspersed with a washout period between diets. A total of 10 healthy, moderately
hypercholesterolaemic, male volunteers consumed the two diets. The project was approved
by the Clinical Research Ethical Committee of the Hospital Universitari de Sant Joan, Reus.
Instrumentation: kinetic studies were performed at the end of each diet using a 16h primed
constant infusion of stable isotope
2
H3-L-leucine. Lipoprotein fractions were separated using
ultracentrifugation technique. Stable isotope incorporated into proteins was measured using
GC-MS. The data obtained were analysed applying multi-compartmental modelling technique
with the SAAM II program. ApoA-I and A-II kinetic studies were conducted in our Research
Unit in Reus. Apo B-100 kinetic studies and kinetic parameter modelling were performed in
collaboration with Dr. Caslake and Professor Packard of the Vascular Biochemistry Section,
Division of Cardiovascular & Medical Sciences, Royal Infirmary, University of Glasgow,
Glasgow, Scotland. Results: the Mediterranean diet, compared to the STEP II diet,
significantly increases apoA-I plasma concentrations. A total of 17 kinetic studies have been
performed but, due to methodological complexity, only the results of 7 kinetic studies (4
following a Mediterranean diet and 3 following a STEP II diet) that have been analysed in a
GC-MS to-date, are presented in this thesis. Despite the limitation of the low number of
kinetic studies analysed, we are able to document that the Mediterranean diet induces a high
apoA-I HDL production rate compared to the STEP II diet. Also, the Mediterranean diet
induces a high apo B-100 LDL production rate and fractional catabolic rate compared to the
STEP II diet. Conclusions: we have, for the first time in Spain, implemented the necessary
methodology to perform apo B-100, apoA-I and A-II kinetic studies in vivo using stable
isotopes in human subjects. A high apoA-I production rate is the main determinant of high
plasma concentrations of apoA-I, and not variations in its catabolism. Lipoprotein kinetic
studies enable the monitoring of lipoprotein metabolic parameters and the investigation of
nutritional and pharmacologic interventions in the primary and secondary prevention of
cardiovascular disease targets.
Els efectes antiaterogènics atribuïts a una dieta de tipus mediterrani, rica en àcids grassos
monoinsaturats (AGM), aportats per oli d'oliva verge, són deguts, en part, a l'augment o al
manteniment de les concentracions plasmàtiques de colesterol de les lipoproteïnes d'alta
densitat (HDL). No obstant i fins al moment, no es coneixen del tot els mecanismes que
expliquen aquestes concentracions. L'apolipoproteïna (apo) A-I (apoA-I) és l'apolipoproteïna
majoritària de les HDL i els seus paràmetres cinètics, com ara la taxa de producció i la taxa
de catabolisme, reflexen la cinètica de les partícules d'HDL. La nostra hipòtesi de treball és la
següent: una dieta Mediterrània rica en AGM aportats per oli d'oliva verge, comparada amb
una dieta STEP II, incrementa o manté els nivells de colesterol de les HDL degut a un
augment de la producció i no a una disminució de la degradació d'apoA-I. Possiblement, la
realització d'estudis de cinètiques utilitzant isòtops estables és la forma més fisiològica
d'avaluar la producció i la degradació d'apolipoproteïnes en l'home. Objectiu: Implementar la
metodologia necessària per realitzar cinètiques d'apo B-100 i, especialment, d'apoA-I i
d'apoA-II en voluntaris in vivo utilitzant isòtops estables com a marcatge de les proteïnes.
Aplicar aquesta metodologia per comparar els efectes d'una dieta Mediterrània rica en oli
d'oliva verge amb una dieta pobra en greixos STEP II sobre el metabolisme de les HDL i de
les lipoproteïnes de baixa densitat (LDL). Disseny: Estudi creuat randomitzat de 4 setmanes
d'intervenció dietètica, amb un període de rentat entre elles. 10 voluntaris, homes sans,
moderadament hipercolesterolèmics van seguir ambdues dietes. Aquest estudi va ser
aprovat pel Comitè d'Ètica i d'Investigació Clínica de l'Hospital Universitari de Sant Joan de
Reus. Instrumentalització: Estudi cinètic mitjançant la injecció i perfusió d'isòtop estable 2H3-
L-leucina en forma d'un bolus inicial i de perfusió durant 16h al final de cada dieta. Les
diferents fraccions lipoproteïques es van separar per ultracentrifugació. La detecció d'isòtop
incorporat va ser mitjançant GC-MS. Les dades obtingudes es van analitzar amb models
multicompartimentals i el programa SAAM II. Els estudis cinètics d'apoA-I i d'apoA-II es van
posat a punt i realitzat a la nostra Unitat de Recerca a Reus. Els estudis d'apo B-100 i la
modelització de les dades cinètiques es van realitzar en colClaboració amb el grup de recerca
de la Vascular Biochemistry Section, Division of Cardiovascular & Medical Sciences, Royal
Infirmary, University of Glasgow, Glasgow). Resultats: La dieta Mediterrània, comparada
amb la dieta STEP II, incrementa significativament la concentració plasmàtica d'apoA-I.
S'han realizat 17 cinètiques però, degut a la complexitat de la metodologia, en aquesta tesis
es presenten els resultats de les 7 cinètiques (4 després de la dieta Mediterrània i 3 després
de la dieta STEP II) analitzades per GC-MS fins ara. A pesar de l'escàs nombre de cinètiques,
s'observen certes tendències. La dieta Mediterrània, comparada amb la dieta STEP II,
presenta una major taxa de producció d'apoA-I de les HDL, així com una major taxa de
producció i de catabolisme de l'apo B-100 de les LDL. Conclusions: S'ha implementat la
metodologia dels estudis de cinètiques amb isòtops estables de l'apoA-I i l'apoA-II de les
HDL i de l'apo B-100 de les VLDL1, VLDL2, IDL i LDL. El determinant de les concentracions
més elevades d'apoA-I en plasma és una major taxa de producció d'aquesta apo i no
diferències en el seu catabolisme. Les aportacions de las cinètiques de les lipoproteïnes
permetran avançar en els mecanismes lipídics involucrats en les malalties vasculars i aportar
nous aspectes sobre les dianes nutricionals i farmacològiques.
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Wu, Guanmin. "Synthesis, characterization, and kinetics of isomerization, C-H and P-C bond activation for unsaturated diphosphine-coordinated triosmium carbonyl clusters." Thesis, University of North Texas, 2008. https://digital.library.unt.edu/ark:/67531/metadc6037/.
Full textAbstract:
Substitution of MeCN ligands in the activated cluster Os3(CO)10(MeCN)2 by the unsaturated diphosphine ligands (Z)-Ph2PCH=CHPPh2 (cDPPEn) or 4,5-bis(diphenylphosphino)-4-cyclopenten-1,3-dione (bpcd) proceeds rapidly at room temperature to furnish the ligand-bridged cluster 1,2-Os3(CO)10(P-P) (P-P represents cDPPEn or bpcd). Heating 1,2-Os3(CO)10(P-P) leads to the formation of the thermodynamically more stable chelating isomer 1,1-Os3(CO)10(P-P). Each compound of Os3(CO)10(P-P) has been characterized by x-ray diffraction, IR, 31P NMR and 1H NMR. Ligand isomerization kinetics have been investigated by UV-VIS and 31P NMR (for cDPPEn) or 1H NMR (for bpcd) spectroscopies. The isomerization mechanism is discussed based on the activation parameters and CO inhibition (for cDPPEn) or ligand trapping experiments (for bpcd). Thermolysis of 1,1-Os3(CO)10(bpcd) in refluxing toluene gives the hydrido cluster HOs3(CO)9[μ-(PPh2)C=C{PPh(C6H4)}C(O)CH2C(O)] and the benzyne cluster HOs3(CO)8(μ3-C6H4)[μ2,η1-PPhC=C(PPh2)C(O)CH2C(O)]. Photolysis of 1,1-Os3(CO)10(bpcd) using near UV light affords HOs3(CO)9[μ-(PPh2)C=C{PPh(C6H4)}C(O)CH2C(O)] as the sole product. HOs3(CO)8(μ3-C6H4)[μ2,η1-PPhC=C(PPh2)C(O)CH2C(O)] has been characterized in solution by IR and NMR spectroscopies. Furthermore its molecular structure has been determined by X-ray crystallography. Reversible C-H bond formation in HOs3(CO)9[μ-(PPh2)C=C{PPh(C6H4)}C(O)CH2C(O)] is demonstrated by ligand trapping studies to give 1,1-Os3(CO)9L(bpcd) (where L = CO, phosphine) via the unsaturated intermediate 1,1-Os3(CO)9(bpcd). The kinetics for reductive coupling in HOs3(CO)9[γ-(PPh2)C=C{PPh(C6H4)}C(O)CH2C(O)] and DOs3(CO)9[μ-(PPh2-d10)C=C{P(Ph-d5)(C6D4)}C(O)CH2C(O)] in the presence of PPh3 give rise to a kH/kD value of 0.88, whose magnitude supports the existence of a preequilibrium involving the hydride(deuteride) cluster and a transient arene-bound Os3 species that precedes the rate-limiting formation of 1,1-Os3(CO)9(bpcd). Strong proof for the proposed hydride(deuteride)/arene preequilibrium has been obtained from photochemical studies employing the isotopically labeled cluster 1,1-Os3(CO)10(bpcd-d4ortho), whose bpcd phenyl groups each contain one ortho hydrogen and deuterium atom. Equilibrium and kinetic isotope effects in the orthometallation step has been determined by 1H NMR in photochemical studies. Kinetics for the transformation from HOs3(CO)9[μ-(PPh2)C=C{PPh(C6H4)}C(O)CH2C(O)] to HOs3(CO)8(μ3-C6H4)[μ2,η1-PPhC=C(PPh2)C(O)CH2C(O)] has been studied by UV-VIS spectroscopy for which the mechanism is discussed.
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Francis, Kevin. "On the Biochemistry, Mechanism and Physiological Role of Fungal Nitronate Monooxygenase." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/chemistry_diss/51.
Full textAbstract:
Nitronate monooxygenase (E.C. 1.13.11.16), formerly known as 2-nitropropane dioxygenase (EC 1.13.11.32), is a flavin dependent enzyme that catalyzes the oxidation of nitronates to their corresponding carbonyl compounds and nitrite. Despite the fact that the enzyme was first isolated from Neurospora crassa 60 years ago, the biochemical and physiological properties of nitronate monooxygenase have remained largely elusive. This dissertation will present the work that established both the catalytic mechanism and physiological role of the fungal enzyme.
The biological and biochemical properties of propionate-3-nitronate, the recently discovered physiological substrate for nitronate monooxygenase, will be extensively reviewed. The nitronate is produced by a variety of variety leguminous plants and fungi and is a potent and irreversible inhibitor of succinate dehydrogenase. Nitronate monooxygenase allows N. crassa to overcome the toxicity of propionate-3-nitronate as demonstrated by in vivo studies of the yeast, which showed that the wild-type can grow in the presence of the toxin whereas a knock out mutant that lacks the gene encoding for the enzyme could not.
In addition to establishing the physiological role of nitronate monooxygenase, the work presented here demonstrates that the catalytic mechanism of the enzyme involves the formation of an anionic flavosemiquinone intermediate. This intermediate is stabilized by the protonated form of an active site histidine residue (His-196) that acts as an electrostatic catalyst for the reaction as demonstrated by pH studies of the reductive half reaction of the enzyme. Histidine 196 also serves as the catalytic base for the reaction of the enzyme with nitroethane as substrate as revealed through mutagenesis studies in which the residue was replaced with an asparagine.
The kinetic implications of branching of reaction intermediates in enzymatic catalysis are also demonstrated through studies of the kinetic isotope effects of nitronate monooxygenase with 1,1-[2H2]-nitroethane as substrate. Finally the use of competitive inhibitors as a probe of enzyme structure will be presented through a study of the inhibition of nitronate monooxygenase with mono-valent inorganic ions. The dissertation will close with unpublished work on the enzyme and concluding remarks concerning the biochemistry and physiology of nitronate monooxygenase.
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Juillet, Clara. "Mécanismes et cinétiques de perméation de l’hydrogène dans les alliages de zirconium oxydés Kinetics of hydrogen desorption from Zircaloy-4: Experimental and modelling Effect of a pre-oxidation on the hydrogen desorption from Zircaloy-4 Effect of the precipitates on the hydrogen desorption kinetics from zirconium-niobium alloys Kinetics of deuterium permeation through Zircaloy-4 in the 623 – 773 K temperature range." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPAST060.
Full textAbstract:
Au cœur des réacteurs à eau pressurisée, les pastilles de combustible sont isolées du circuit primaire par des gaines en alliages de zirconium. En conditions nominales de fonctionnement, l’interaction entre ces gaines et le milieu primaire entraîne la formation d’un oxyde et l’absorption d’hydrogène par l’alliage. La désorption de l’hydrogène lors du transport de combustible usé et le relâchement du tritium issu de la réaction de fission au sein des pastilles de combustible en réacteur sont des problématiques importantes pour la sûreté. Il est impératif d’estimer la quantité de tritium susceptible d’être relâchée des assemblages dans le milieu primaire ainsi que les quantités d’hydrogène désorbé sous vide ou gaz neutre pendant le transport. Au-delà de cet aspect quantitatif, comprendre le mécanisme de perméation de l’hydrogène et de ses isotopes dans les gaines en alliage de zirconium est nécessaire si l’on veut maîtriser les rejets et émissions de ces produits et in fine la sûreté. Le processus de perméation du tritium à travers une gaine oxydée peut se décomposer en 9 étapes : 1. Adsorption de tritium à la surface de l’oxyde formé sur la face interne ;2. Passage des sites de surface aux sites de subsurface en tant qu’espèce absorbée ;3. Diffusion de tritium dans l’oxyde jusqu’à l’interface oxyde/métal ;4. Intégration de tritium dans le métal ;5. Diffusion de tritium dans le métal ;6. Intégration de tritium dans l’oxyde ;7. Diffusion de tritium dans l’oxyde jusqu’à la subsurface ;8. Passage des sites de subsurface aux sites de surface en tant qu’espèce adsorbée ;9. Recombinaison et désorption.Les objectifs de l’étude sont de déterminer l’étape cinétiquement limitante du processus de perméation de l’hydrogène dans les alliages Zircaloy-4 et M5Framatome, de quantifier les constantes cinétiques associées et de développer un modèle numérique pouvant prédire les flux de désorption de l’hydrogène. De par la complexité du processus de perméation du tritium à travers une gaine de combustible oxydé, un système simplifié sans couche d’oxyde a initialement été étudié. Le processus de désorption de l’hydrogène a été investigué par thermodésorption en rampe de température ainsi qu’en isotherme, et modélisé par éléments finis avec le code Cast3M. Les constantes cinétiques ont été évaluées et optimisées en couplant le modèle Cast3M et l’outil d’optimisation de la plateforme URANIE. Le flux de désorption s’est avéré être proportionnel au carré de la concentration en hydrogène avec une constante de désorption dont l’énergie d’activation est égale à 290 ± 10 kJ/mol. Pour le M5Framatome, les conclusions et résultats sont quasi-identiques au cas précédent, à ceci près que la désorption de l’hydrogène piégé par les précipités riches en niobium lors de la fabrication semble être assujettie à leur dissolution. Le développement d’un banc de perméation de deutérium gazeux fonctionnant entre 623 et 773 K a permis de mettre en évidence que la cinétique du processus de perméation est également limitée par l’étape de recombinaison de surface pour le Zircaloy-4. Ces essais de perméation ont révélé le rôle important de l’état de surface, puisque l’application par le dispositif d’une contrainte et d’une déformation induit une augmentation du flux de désorption. Des essais d’oxydation ont ensuite été effectués sous diverses atmosphères. Lors d’essai de désorption en rampe de température, la dissolution de l’oxyde dans le métal semble piloter la cinétique de désorption de l’hydrogène. Le développement de modèles numériques et analytiques a permis de démontrer dans le cas d’une dissolution négligeable de la zircone, une cinétique de désorption contrôlée par un régime mixte de diffusion de l’hydrogène dans l’oxyde et de recombinaison surfacique et de quantifier la constante de désorption et le coefficient de diffusion dans la zircone.A partir des résultats de la thèse, une estimation de la quantité de tritium relâché sous vide a pu être établieAt the heart of Pressurized Water Reactors, the uranium oxide fuel leads isolated from the primary circuit by zirconium alloys cladding. Under normal operating conditions, the interaction between these claddings and the primary water results in the formation of an oxide scale and the hydrogen absorption in the alloy. During operation in a reactor and during the transport of used nuclear fuels, the desorption of this hydrogen and the tritium resulting from the ternary fission from the fuel rods is a safety concern. It is imperative to estimate the amount of tritium prone to be released from the fuel assemblies into the primary water and the quantities of hydrogen desorbed under vacuum or neutral gas for the transport issue. Beyond this quantitative aspect, understanding the permeation mechanism of hydrogen and its isotopes in zirconium alloy claddings is necessary if we want to control releases and more generally safety. The tritium permeation through the oxidized fuel cladding can be decomposed into 9 steps: 1. Tritium adsorption on the oxide formed on the inner face; 2. Passing from surface sites to subsurface sites as absorbed species; 3. Tritium diffusion through the oxide towards the oxide/metal interface; 4. Integration into the metal; 5.Tritium diffusion through the metal; 6. Integration into the oxide lattice; 7. Tritium diffusion through the oxide towards the subsurface; 8. Passing from subsurface sites to surface sites as adsorbed species; 9. Recombination and desorption.This study is aimed at identifying, among these 9 steps, the kinetically limiting step of the hydrogen permeation process in Zircaloy-4 and M5Framatome alloys, quantifying the associated kinetic constants and developing a numerical model that can predict hydrogen desorption fluxes. Due to the complexity of the tritium permeation process through an oxidized fuel cladding, a simplified system without an oxide layer was initially studied. A study on the hydrogen desorption process was carried out by thermodesorption in temperature ramp and in isotherm, in order to model by finite elements with the Cast3M tool the desorption kinetics and to quantify the associated kinetic constant by coupling with the URANIE optimization tool. The desorption flux was found to be proportional to the square of the hydrogen concentration with a desorption constant whose activation energy is equal to 290 ± 10 kJ/mol. A similar study on M5Framatome revealed during a temperature ramp that hydrogen desorption from niobium-rich precipitates appears to be controlled by their dissolution. The development of a deuterium permeation device operating between 623 and 773 K has demonstrated that the rate-limiting step of the hydrogen permeation is the surface recombination for Zircaloy-4. These permeation experiments have highlighted the important role of surface state, because the application by the device of stress and strain induces a desorption flux increase. Oxidation tests were then carried out under various atmospheres. During a temperature ramp desorption experiment, the oxide dissolution into the metal appears to control the kinetics of hydrogen desorption. The development of numerical and analytical models made it possible to demonstrate in the case of a negligible zirconia dissolution, a desorption kinetics limited by a mixed regime of hydrogen diffusion through the oxide and surface recombination and to quantify the desorption constant and diffusion coefficient in zirconia. From the thesis results, an estimate of the tritium released quantity under vacuum was established
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Bajnai, David [Verfasser], Jens [Gutachter] Fiebig, Silke [Gutachter] Voigt, and Gregory D. [Gutachter] Price. "The clumped and oxygen isotope compositions of biogenic carbonate archives: kinetic effects and reconstruction of seawater temperatures and seawater δ18O / David Bajnai ; Gutachter: Jens Fiebig, Silke Voigt, Gregory D. Price." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2019. http://d-nb.info/1193126037/34.
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Montanari, Carlo Emilio. "Development of an event-based simulator for analysing excluded volume effects in a Brownian gas." Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2017. http://amslaurea.unibo.it/14527/.
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Il presente lavoro si pone come scopo lo sviluppo di un simulatore in C++ di dinamica molecolare utilizzando un approccio event-based, in grado di simulare la dinamica newtoniana semplice di molecole bidimensionali di forma arbitraria. Abbiamo utilizzato il simulatore NOCS per imbastire un primo tentativo di ricerca e di analisi degli effetti di volume escluso sul moto Browniano di molecole. In particolare si vogliono ricercare violazioni locali di isotropia nel moto Browniano. Nella parte teorica dell'elaborato, si analizzano gli strumenti matematici e statistici fondamentali della Kinetic Theory (teoria cinetica dei gas) ed i principali modelli della depletion force, uno dei fenomeni causati da potenziale di volume escluso.
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Hancock, Amber N. "A Radical Approach to Syntheses and Mechanisms." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/77139.
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The critically important nature of radical and radical ion mechanisms in biology and chemistry continues to be recognized as our understanding of these unique transient species grows. The work presented herein demonstrates the versatility of kinetic studies for understanding the elementary chemical reactions of radicals and radical ions.
Chapter 2 discusses the use of direct ultrafast kinetics techniques for investigation of crucially important enzymatic systems; while Chapter 3 demonstrates the value of indirect competition kinetics techniques for development of synthetic methodologies for commercially valuable classes of compounds. The mechanism of decay for aminyl radical cations has received considerable attention because of their suspected role as intermediates in the oxidation of tertiary amines by monoamine oxygenases and the cytochrome P450 family of enzymes. Radical cations are believed to undergo deprotonation as a key step in catalysis. KIE studies performed by previous researchers indicate N,N-dimethylaniline radical cations deprotonate in the presence of the bases acetate and pyridine. By studying the electrochemical kinetics of the reaction of para substituted N,N-dimethylaniline radical cations with acetate anion, we have produced compelling evidence to the contrary. Rather than deprotonation, acetate reacts with N,N-dimethylaniline radical cation by electron transfer, generating the neutral amine and acetoxyl radical.
Transport properties of reactants and solvent polarity changes were investigated and confirmed not to influence the electrochemical behavior forming the basis for our mechanistic hypothesis. To reconcile our conclusion with earlier results, KIEs were reinvestigated electrochemically and by nanosecond laser flash photolysis. Rather than a primary isotope effect (associated with C-H bond cleavage), we believe the observed KIEs are secondary, and can be rationalized on the basis of a quantum effect due to hyperconjugative stabilization in aromatic radical cations during an electron transfer reaction. Product studies performed by constant potential coulometry indicate N,N-dimethylaniline radical cations are catalytic in carboxylate oxidations. Collectively, our results suggest that aminyl radical cation deprotonations may not be as facile as was previously thought, and that in some cases, may not occur at all.
Interest in design and synthesis of selenium containing heterocycles stems from their ability to function as antioxidants, anti-virals, anti-inflammatories, and immunomodulators. To establish synthetic feasibility of intramolecular homolytic substitution at selenium for preparation of selenocycles, we set out to determine what factors influence cyclization kinetics.
A series of photochemically labile Barton and Kim esters have been syntheisized and employed as radical precursors. The effect of leaving radical stability on kinetics has been investigated through determination of rate constants and activation parameters for intramolecular homolytic substitution of the corresponding radicals via competition experiments. Notable leaving group effects on measured kinetic parameters show more facile reactions for radical precursors with more stable leaving radicals. Moreover, cyclizations to form six-membered (as opposed to five- membered) ring systems exhibited order of magnitude decreases in rate constants for a given leaving radical. Our results are congruent with expectations for radical cyclizations trends for the varied experimental parameters and suggest homolytic substitution affords a convenient means for synthesis of selenocycles.Ph. D.