Browsing by Subject "Enzyme"

Now showing 1 - 5 of 5
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    Computational Studies of the Chemical Properties of Complex Metalloenzyme Systems and Transition Metal Catalysis using Electronic Structure Methods
    (2021-06) McGreal, Meghan
    Computational chemistry is a useful tool for studying the aspects of chemistry that cannot be wholly studied in a laboratory setting, such as mechanisms, electronic structure, reaction barriers, and other various chemical and physical properties. In this dissertation, computational chemistry methods, specifically Density Functional Theory (DFT), is utilized to study complex catalytic systems. These systems include transition-metal based metalloenzymes and molecular catalysts that were studied utilizing electronic structure methods. Chapters 2, 3, and 4 focus on elucidation of catalysis, structural features, function, and other various properties of the [NiFe]-hydrogenase enzyme system. Specifically, the mechanism of catalysis was studied, appropriate models for the study of this bimetallic enzyme active site were determined (Chapter 2), the effect of mutation of highly conserved residues were analyzed (Chapter 3), and the influence biomimetic-inspired changes to the active site were assessed (Chapter 4). Chapter 5 focuses on elucidating the structure and function of the non-heme Fe chlorination enzyme, SyrB2, in collaboration with the Bhagi-Damodaran Group. Chapter 6 is a study of titanium-catalyzed nitrene transfer of diazenes to isocyanides for carbodiimide synthesis in collaboration with the Tonks Group. Finally, Chapter 7 is computational determination of the chemical hardness of various anions, as well as the effect of stability of anion impact, selectivity and affinity for Gd-containing complexes in collaboration with the Pierre Group.
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    Enzyme catalyzed perhydrolysis, molecular basis and application
    (2011-10) Yin, Delu (Tyler)
    Enzyme catalyzed perhydrolysis converts a carboxylic acid or ester to a peracid. In the former reaction, the amount of peracid generated is thermodynamically controlled (Keq = 3) – while in the latter, the reaction is kinetically controlled, thus a higher concentration of peracid can be generated. Enzymes that catalyze perhydrolysis of carboxylic acids share high sequence similarity and are thought to use an esterase-like mechanism. Alternatively, carboxylic acids can also use a non-covalent mechanism, such as those used by hydroxynitrile lyases. To test whether carboxylic acid perhydrolases use an esterase-like mechanism, we identify a key covalent intermediate by mass spectrometry that can be attributed to an esterase-like mechanism but not a non-covalent mechanism. We also find that carboxylic acid perhydrolases are good catalysts for hydrolysis of peracetic acid, suggesting that their natural role is to degrade peracids generated as by-products in a living organism. Next, we determine how perhydrolases increase the rate of perhydrolysis. Carboxylic acid perhydrolases increase the rate of perhydrolysis by either increasing the selectivity for hydrogen peroxide or lower the activation barrier towards acylenzyme formation. We measure the selectivity of hydrogen peroxide using wild-type Pseudomonas fluorescens esterase (PFE) and L29P PFE (a model carboxylic acid perhydrolase). The L29P PFE variant is less selective for hydrogen peroxide than the wild-type despite having higher perhydrolysis activity. We measure the rate of acyl-enzyme formation using isotope exchange of acetic acid in H218O/H216O. The L29P PFE variant catalyzes the isotope exchange rate faster than the wild-type. Thus, carboxylic acid perhydrolases favor the formation of acyl-enzyme from carboxylic acids. We find that carboxylic acid perhydrolase (L29P PFE) does not catalyze ester perhydrolysis for accumulating high concentrations of peracetic acid. Instead, wild-type PFE and a new variant, F162L PFE accumulate up to 130 mM of peracetic acid. We measure kinetic parameters and show that hydrolysis of peracetic acid limits maximum accumulation. The F162L PFE variant minimizes hydrolysis of peracetic acid by lower ing the Km and increasing the kcat for ethyl acetate hydrolysis. The kinetic parameters are also used to predict the maximum amount of peracetic acid that can be accumulated. The F162L PFE variant is used to improve the efficiency of lignocellulose pretreatment from a previously published result using wild-type PFE. Enzymatically generated peracetic acid reacts converts lignin into smaller and more soluble lignin pieces. The chemoenzymatic process is further improved by forming peracetic acid in a biphasic layer which allows the reuse of enzyme. The pretreatment reaction conditions were also optimized by increasing the temperature to 60 °C and reducing the reaction time to 6 hours.
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    Intensified biocatalysis for production of fuel and chemicals from lipids.
    (2010-03) Zhao, Xueyan
    Triglycerides are abundant biorenewable resources found in vegetable oils and fats. The effective utilization of triglycerides is one of the key interests in developing renewable fuels and products. However, triglycerides are difficult and inefficient to be used as fuels directly in regular combustion engines. The area of biodiesel synthesis concerns reactions converting triglycerides to methyl or ethyl monoesters for better fuel properties. This process releases glycerol as byproduct. This thesis aims at developing novel biocatalytic conversion of triglycerides and glycerol for the production of fuels and chemicals. One key challenge in realizing efficient biocatalytic synthesis of biodiesel is to improve reaction velocity and catalyst efficiency. This research explored a unique approach by developing organic-soluble lipase for a one-pot synthesis-and-use strategy. The productivities of the modified lipase in a water-free reaction system were found to be over two orders of magnitude higher than previously reported results. Whereas native lipases showed no activity in the absence of water, the organic soluble lipase demonstrated reaction rates of up to 33 g-product/g-enzyme-h. As for the byproduct (glycerol) from biodiesel synthesis, current research has mostly focused on derivation of value-added chemicals instead of being used as a simple additive in processing of food and personal care products. Key issues centered on how to produce the desired products most efficiently and selectively from glycerol. Enzymatic conversion of glycerol can produce 1,3-dihydroxyacetone (DHA), a unique and versatile chemical with a broad range of application potentials, by selective oxidizing the hydroxyl group(s) of glycerol. The price of DHA is more than 200 times higher than that of glycerol. In this work, DHA production was tackled through a novel biocatalytic process. Focus was placed on several aspects in understanding and optimizing the process including selection and improvement of biocatalyst, development of novel carbon electrode materials for cofactor regeneration, and reactor design. One potential advantage of using bioelectrochemical method for cofactor regeneration is the possibility to integrate the biochemical process with biofuel cells for simultaneous chemical production and power generation. Toward that, this thesis explored the necessary fundamental issues, including the construction and study of a model glucose/oxygen biofuel cell.
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    Reactions of copper complexes with dioxygen and oxo transfer reagents: toward elusive copper-oxyl species.
    (2010-06) Hong, Sungjun
    The binding and activation of dioxygen by Cu ions is central to the function of numerous biological systems. Among the enzymes activate dioxygen for the functionalization of organic substrates, those catalyzed by the mononuclear copper enzymes dopamine β-monooxygenase (DβM) and peptidylglycine α-hydroxylating monooxygenase (PHM) are less understood. Despite extensive research on these enzymes, the exact nature of the active species responsible for substrate functionalization is not resolved, with two provocative proposals involving either a CuII-superoxo or a mononuclear CuII-oxyl species having been put forth. The goal of this research is to understand the reaction catalyzed by the PHM and DβM enzymes on a fundamental chemical level via a small molecule synthetic model approach, with particular emphasis on generating and/or characterizing a Cu-oxygen species that is capable of performing similar reactions to those seen in the DβM and PHM enzymes. Chapter 1 contains a general overview of dioxygen activation in biological systems and gives a review of the structure and proposed catalytic mechanisms of DβM and PHM, followed by a summary of recent synthetic efforts toward mononuclear Cu/O2 adducts and Cu-oxyl species. Chapter 2 describes the synthesis and characterization of the copper(I) complexes of the electron-deficient β-diketiminate and analogous 4- nitroformazan supporting ligands, and their O2-reactivity studies, portions of which have been previously reported.1 Chapter 3 describes a bio-inspired synthetic route toward a mononuclear Cu-oxyl species that involves decarboxylation of copper(I)-α- ketocarboxylate complexes by dioxygen; portions of the work have been communicated previously.2 Chapter 4 then describes results obtained from reactions of copper(I) complexes of bidentate N-donor ligands with pyridine- and trimethylamine N-oxides or PhIO. Portions of this work were previously reported.3
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    Self-assembling Phosphoramidate Pronucleotides: Enzymatic Regulation and Application Towards Therapeutic Delivery
    (2019-08) West, Harrison
    Prior characterizations of the nucleoside phosphoramidate moiety have centered upon the ability of amine containing side-chains to mask the negative charge inherent to monophosphorylated nucleosides for the purpose of enhancing their passive movement across biological membranes. When used for the intracellular delivery of therapeutic nucleoside monophosphate analogs, these molecules are referred to as phosphoramidate “ProTides” and represent an important class of antiviral and anticancer prodrugs. The primary aim of this thesis to build upon these works and present the nucleoside phosphoramidate moiety as a multifunctional regulator of molecular self-assembly. Appendage of nucleoside phosphoramidates to molecules such as self-assembling peptides was shown to modulate the self-assembling properties of the molecules through alteration of the non-covalent interactions between individual monomers and nanostructure assemblies. Additionally, the nucleoside phosphoramidate moiety was found to impart enzyme responsive qualities. Histidine triad nucleotide binding proteins (Hints), an enzyme class that possesses phosphoramidase activity, were found to regulate the assembly of nucleoside phosphoramidate bearing nanostructures by inducing ionic interaction mediated crosslinking after enzymatic hydrolysis. Chemical modification of self-assembling peptides with nucleoside phosphoramidates bearing non-natural and therapeutic nucleosides was also achieved to effect the first ever demonstrated self-assembling phosphoramidate ProTides as one-component nanomedicines. The developed formulations are currently under investigation for localized delivery of cancer chemotherapeutic prodrugs