Biocatalysis has become a popular technique in organic synthesis due to high activity and selectivity of enzyme catalyzed reactions. Enantioselectivity is a particularly attractive enzyme property, which is utilized for the production of enantiopure substances. Determination of the temperature dependence of enzyme enantioselectivity allows for thermodynamic analyses that reveal the contribution of differential activation enthalpy, ΔR-SΔH‡, and entropy, ΔR-SΔS‡. In the present investigation the influence of substrate structure, variations on enzyme structure and of reaction media on the enantioselectivity of Candida Antarctica lipase B has been studied. The contribution of enthalpy, ΔR-SΔH‡, and entropy, TΔR-SΔS‡, to the differential free energy, ΔR-SΔG‡, of kinetic resolutions of sec-alcohols were of similar magnitude. Generally the two terms were counteracting, meaning that the enantiomer favored by enthalpy was disfavored by entropy. 3-Hexanol was an exception where the preferred enantiomer was favored both by enthalpy and by entropy. Resolution of 1-bromo-2-butanol revealed non-steric interactions to influence both ΔR-SΔH‡ and ΔR-SΔS‡. Molecular modeling of the spatial freedom of the enzyme-substrate transition state indicated correlation tothe transition state entropy. The acyl chain length was shown to affect enantioselectivity in transesterifications of a sec-alcohol. Point mutations in the active site were found to decrease or increase enantioselectivity. The changes were caused by partly compensatory changes in both ΔR-SΔH‡ and ΔR-SΔS‡. Studies on single and double mutation variants showed that the observed changes were not additive. Enantioselectivity was strongly affected by the reaction media. Transesterifications of a sec-alcohol catalyzed by Candida Antarctica lipase B was studied in eight liquidorganic solvents and supercritical carbon dioxide. A correlation of enantioselectivity and the molecular volume of the solvent was found. Differential activation enthalpy, ΔR-SΔH‡, and entropy, ΔR-SΔS‡, display a compensatory nature. However this compensation is not perfect, which allows for modifications of enantioselectivity. The components of the thermodynamic parameters are highly complex and interdependent but if their roles are elucidated rational design of enantioselective enzymatic processes may be possible. / QC 20100616

Caspase-3 is a cysteine protease that hydrolyzes diverse intracellular proteins during programmed cell death (known as apoptosis). It has been a popular target for drug design against abnormal cell death for more than a decade. No approved caspase based drug, however, is available so far. Therefore, structural insights about the substrate recognition of caspase-3 are needed for the future development of caspase-3 based inhibitors and drugs. In this study, crystal structures of recombinant caspase-3 in complex with seven substrate analog inhibitors, including acetyl (Ac)-DEVD-aldehyde (Cho), Ac-DMQD-Cho, Ac-IEPD-Cho, Ac-YVAD-Cho, Ac-WEHD-Cho, Ac-VDVAD-Cho, and tert-butoxycarbonyl (Boc)-D-fluoromethylketone (Fmk), have been analyzed in combination with enzyme kinetic data and computational models. Seven crystal structures were determined at resolutions of 1.7-2.6Å. The binding conformation of each inhibitor residue at P1-P4 position was analyzed. The negative P1 aspartic acid side chain is exclusively required by the positive S1 pocket of caspase-3. Small hydrophobic P2 residues are preferred by the nonpolar S2 pocket formed by Y204, W206, and F256. Although hydrophilic residues at P3 position tend to fit better, hydrophobic residues also can be accommodated by the plastic S3 pocket. Two substrate binding sites were found in the S4 pocket, one formed by main chain atoms of F250 and side chain atoms of N208 and the other formed by aromatic side chains of W206 and W214. These two binding sites are responsible for the binding of hydrophilic and hydrophobic P4 residues, respectively. Furthermore, the S5 subsite of caspase-3 formed by side chains of F250 and F252 was discovered. It stabilizes hydrophobic P5 residues on the substrates by an induced fit mechanism. Computational studies were performed to help improve prediction of protein structures and protein-ligand interactions. Based on the Morse’s function, a novel potential function with only three adjustable parameters per residue pair was developed, which will significantly increase the efficiency of protein structure prediction and molecular mechanics. Altogether, our studies have provided valuable information for the future caspase-3 based drug development.

A dinâmica estrutural fundamentando a função das xilanases GH11 ainda não está clara. Novo conhecimento sobre a dinâmica catalítica dessas enzimas é crucial para a engenharia de novas enzimas melhoradas beneficiando, assim, diversas indústrias biotecnológicas e de química verde. Com base nesse fato, esse trabalho teve por objetivo obter novas informações acerca da dinâmica catalítica de uma xilanase GH11, através do uso de um conjunto de diversas técnicas avançadas de biofísica molecular em nível bulk e em nível de molécula única (inglês single molecule ou sm). Para isso, foram projetadas xilanases GH11 de Bacillus subtilis ssp. subtilis 168 (XynA) com mutações únicas de cisteína para a marcação dos resíduos D119 e R122 no domínio polegar, do resíduo N54 no domínio dedos, e do resíduo N151 na alfa-hélice, seguidas pela sua construção e produção por métodos de biologia molecular. Esses mutantes foram marcados em seus respectivos grupos tióis com a sonda fluorescente sensível à polaridade Acrylodan, com a sonda de spin MTSSL, e com a sonda fluorescente fotoestável AttoOxa11. A xilanase tipo selvagem for marcada em seu N-terminal com a sonda fotoestável Alexa Fluor® 488 5-SDP Ester. Foram utilizados ensaios de espectrofotometria de fluorescência em nível bulk e de espectroscopia de ressonância paramagnética eletrônica para investigar como a dinâmica do domínio polegar da xilanase GH11, temperatura, e ligação ao substrato se correlacionam um com o outro. Os resultados atestaram que um estado do domínio polegar controlado por temperatura, aberto, dinâmico e flexível tem mais chances de se ligar efetivamente ao substrato de uma maneira produtiva, o que está em completo acordo com estudos anteriores de simulação de dinâmica molecular, cristalografia, desnaturação térmica, e análise funcional por desenho racional de mutantes de domínio polegar de xilanases GH11. Com base nas evidências adquiridas e em estudos anteriores, nós propomos um conjunto de hipóteses e modelos para a dinâmica catalítica da xilanase, focando no papel do domínio polegar nesse processo. No intuito de determinar a constante de afinidade da xilanase por seu substrato e os tempos de relaxamento e constantes de velocidade dos movimentos do domínio polegar, foram feitas medidas de espectroscopia de correlação de fluorescência simples e combinada com transferência eletrônica fotoinduzida, usando as xilanases marcadas com as sondas fluorescentes fotoestáveis, na presença e na ausência de substrato. Os resultados mostraram tempos de difusão muito maiores para as xilanases na presença de substrato, como efeito da afinidade da enzima pelo mesmo. Entretanto, não foi verificada nenhuma curva de decaimento como efeito de supressão dinâmica da sonda por PET. Esses mesmos conjugados foram aplicados com sucesso em microscopia por imagem de tempo de vida de fluorescência, no intuito de analisar sistematicamente a afinidade da xilanase por partículas insolúveis e filmes de substrato, e por fragmentos insolúveis de frações de processos de deslignificação e desestruturação de bagaço de cana-de-açúcar, assim como para a análise da composição, estrutura e topologia desses materiais. Foi possível verificar a presença de xilano na maioria das frações desse bagaço tratado, mas em quantidades variáveis / The structural dynamics underlying the function of GH11 xylanases is still unclear. New insights into the catalytic dynamics of these enzymes are crucial for engineering novel improved enzymes benefiting biotechnological and green chemistry industries. The objective of this work was to obtain new information concerning the catalytic dynamics of a GH11 xylanase, by using a combination of advanced molecular biophysics techniques, both at the bulk level and at the single molecule level (sm). Mutant GH11 xylanases from Bacillus subtilis ssp. subtilis 168 (XynA) were designed with single point cysteine mutations for labeling the residues D119 and R122 on the thumb domain, N54 on the fingers domain, and N151 on the alpha helix, followed by their construction and production by molecular biology methods. These mutants were labeled at their respective thiol groups by the polarity sensitive fluorescent probe Acrylodan, by the electron spin probe MTSSL, and by the photostable fluorescent probe AttoOxa11. The wild-type xylanase was labeled at its N-terminus by the photostable fluorescent probe Alexa Fluor® 488 5-SDP Ester. Bulk fluorescence spectrophotometry and electron paramagnetic resonance assays were used to investigate how the thumb domain dynamics of the GH11 xylanase, temperature and substrate binding were correlated. These results demonstrated that a temperature controlled, open, dynamical and flexible thumb domain state is more likely to effectively bind the substrate in a productive way, which is in complete agreement with previous studies from molecular dynamics simulations, crystallography, thermal denaturation, and function analysis by the rational design of thumb mutants for GH11 xylanases. Based on this evidence and previous studies, we proposed a hypothesis for the xylanase catalytic dynamics, focusing on the role of the thumb domain. In order to determine the xylanase affinity constant for its substrate and the relaxation times and rate constants of the thumb domain movements, fluorescence correlation spectroscopy measurements were performed. Both simple and combined measurements with photoinduced electron transfer were performed, using the xylanases labeled with photostable fluorescent probes, in the presence and absence of substrate. The results have shown longer diffusion times for the xylanases in the presence of substrate, as an effect of the enzyme affinity for it. However, it was not verified any decay curve as an effect of the dynamic suppression of the probe via PET. The same conjugates were successfully applied to fluorescence-lifetime imaging microscopy, aiming to systematically analyze the affinity for xylanase of substrates in the form of insoluble particles and films, and for water insoluble fractions from sugarcane bagasse delignification processes. In addition, the composition, structure and topology of these materials was examined. It was possible to verify the presence of xylan in most fractions of this treated bagasse, although in variable quantities

Engineering artificial cells is currently an emerging area of research that involves constructing mimics of biological cells. These biomimetic cellular systems hold tremendous promise for the different biomedical applications (diagnostics, therapy, tissue engineering, gene transfection, bioactive coatings) as well as aspects of synthetic biology. A key architectural principle of the cell is a multicompartmentalized assembly, which is one of the features of biological cells that enable the performance of multiple complex biochemical reactions within confined environments. For this purpose, this study demonstrates novel artificial cells, not only presenting organelle mimics but also incorporating various stimuli for regulating enzymatic cascade reactions within the artificial cell and for controlled simultaneous and/or subsequent release of the encapsulated (therapeutic) molecules. To successfully fabricate the multifunctional polymeric multicompartmentalized systems as artificial cells aimed for, in the first step a hollow capsule as biomimetic cellular membrane was developed to simulate a key characteristic of functional artificial cells for the selective uptake and release of (bio)molecules and particles for intra- and intercellular signaling processes. Herein using LbL technique which involved alternate deposition of oppositely charged polyelectrolytes on silica template via electrostatic interaction, the pH and temperature dual-responsive and photo-crosslinked hollow capsule was fabricated and they can be used for the subsequent post-encapsulation process of protein-like macromolecules (≤ 11 nm) and their controllable release triggered by external stimuli for mimicking the controllable bio-inspired functions of cell membranes. The reversible temperature and pH dual-response ability of the hollow capsules has been analyzed. The uptake and release properties of the resulting hollow capsules with different degree of photo-crosslinking for cargos have been further investigated at various temperatures (25, 37 or 45°C) and pH (5.5 or 7.4) of the solution. Next, the design of the polymersomal subcompartmens as organelle mimics, which divide the interior of the multicompartmentalized systems into subcompartments and can stably encapsulate fragile hydrophobic and hydrophilic cargo, e.g., enzymes in order to conduct encapsulated catalysis-resembling cell organelles, was also an important subject. The fabrication of these subcompartments was starting with the synthesis of suitably end-group block copolymers to realize the enzyme-loaded, multifunctional, pH-responsive, photo-crosslinked and post-labelled polymersomes decorated with adamantane groups. The pH sensitivity and various enzymatic reactions of the established multifunctional Ada-polymersomes have been investigated. Based on the above concepts, a bottom-up approach was developed to assemble a structural and functional eukaryotic cell mimics, including “membrane-associated” multicompartmentalized system (MS1) and “free-floating” multicompartmentalized system (MS2), by loading pH-sensitive Ada-polymersomes inside the multifunctional cell membrane. The creation of these multicompartmentalized systems was based on the assembly of enzyme-loaded Ada-polymersomes as organelle mimics onto sacrificial particle templates by host-guest interaction, followed by the LbL deposition of temperature-responsive and photo-crosslinkable PMA(β-CD)/[PAH/PNMD]3 multilayers and outer protective capping PAH/PMA(PEG) bilayer as biomimetic cellular membrane. Upon photo-crosslinking the polymer biomimetic membrane and dissolution of the particle templates, multicompartmentalized systems were obtained. Spatial position of the subcompartments can be controlled using non-covalent host-guest concept, which yielded multicompartmentalized systems containing “membrane-associated” and “free-floating” subunits. Moreover, the metabolism mimicry of multicompartmentalized systems by performing multiple successive two-enzyme cascade reactions in the cells and the multiple parallel reactions by using a third enzyme for deactivating the reaction product and interfering the cascade reaction have been investigated. In conclusion, these multicompartmentalized systems, combining the advantages of both pH-responsive Ada-polymersomes as organelle mimics and multifunctional hollow capsule as biomimetic cellular membrane, present new opportunities for the development of functional cell mimics. The presented studies highlight crucial aspects for the successful applications of such cell mimics for diagnostics, tissue engineering, as nanoreactors, as carriers for multiple drug delivery with controlled release profiles, or as therapeutic artificial cells.

Les biomolécules sont naturellement immergées dans l’eau, qui joue un rôle clé dans de nombreux processus biologiques. Réciproquement, les propriétés de l’eau sont affectées par la présence de la biomolécule. Dans cette thèse, nous combinons modèles théoriques et simulations numériques pour obtenir une description à l’échelle moléculaire des interactions entre une biomolécule et son environnement. Le manuscrit est structuré en deux parties, abordant deux aspects complémentaires de cette interaction complexe. La première partie est consacrée à la perturbation induite par une biomolécule sur l’eau. Nous déterminons en quoi la couche d’hydratation diffère de l’eau bulk et identifions les facteurs moléculaires en jeu. Nous comparons ensuite les couches d’hydratation d’une protéine antigel et d’une protéine modèle afin de déterminer si les propriétés d’hydratation peuvent expliquer l’activité antigel. Nous étudions enfin la dynamique d’hydratation de l’ADN. Nous obtenons une image résolue spatialement des propriétés de sa couche d’hydratation et y caractérisons les différentes sources d’hétérogénéité. La deuxième partie s’intéresse au rôle de l’environnement sur la catalyse enzymatique. Nous étudions deux systèmes distincts, avec des questions différentes mais une même méthodologie. Nous examinons d’abord le rôle de résidus dans le site actif de la dihydrofolate réductase et obtenons une interprétation moléculaire de résultats expérimentaux récents. Enfin, nous nous intéressons à la catalyse enzymatique en solvant organique, où l’addition de petites quantités d’eau permet d’accélérer la réaction. Nous recherchons une description à l’échelle moléculaire de cet effet. / Biomolecules are immersed in an aqueous solvent, which plays a key role in a wide range of biochemical processes. In addition, the properties of water molecules in the hydration shell are perturbed by the presence of the biomolecule. In this thesis, we combine theoretical models and numerical simulations to provide a molecular description of the interplay between a biomolecule and its environment. The manuscript is structured in two parts, addressing two complementary aspects of this complex interaction. In the first part we focus on the perturbation induced by a biomolecule on water molecules. We determine how much the hydration shell differs from bulk water and we identify the molecular factors at play. We then compare the hydration shells of an antifreeze protein and of a typical protein and investigate whether the shell structure and dynamics can explain the antifreeze properties. We finally study the hydration dynamics of a DNA dodecamer where slow water dynamics was suggested. We obtain a spatially resolved picture of DNA hydration and investigate the sources of heterogeneity. In the second part we examine the role of the environment in the chemical step of enzyme catalysis. We focus on two distinct systems with different questions, but relying on a common simulation methodology. We first examine the role of specific active site residues in catalysis by dihydrofolate reductase and we provide a molecular interpretation of recent experimental results. We finally study the role of water in enzyme catalysis in organic solvents, where addition of small amounts of water was shown to accelerate the chemical step. We seek a molecular scale description of this effect.

<p dir="ltr">Scientists across many disciplines have observed a striking phenomenon: chemical reactions that do not appreciably occur in large volumes often proceed readily in microdroplets. At the core of suggested mechanisms is the influence of interfacial chemistry on the overall reaction; when the interfacial area dominates the reactor volume, the measured reaction rate is often accelerated. For instance, microdroplets with a high surface area-to-volume ratio (generally with radii smaller than 10 µm) provide a unique reaction environment and have been observed to accelerate a wide variety of chemical reactions. This is likely surprising to most readers, as much of our chemical intuition comes from experiments performed on benchtops in beakers (large, single-phase systems). However, microdroplets are regularly exploited by nature, from multiphase atmospheric aerosols to biomolecular condensates in cells. Thus, it is vital to have measurement tools capable of studying multiphase, nanoscale reactors. An electrochemical perspective on measuring multiphase chemistry under nanoconfinement is given in Chapters 2-4. To my knowledge, there were no reports of accelerated reactivity in microdroplets from electrochemical studies until the 2021 observation that enzyme turnover rates are inversely-related to the size of the containing nanodroplet (given in Chapter 6). In this dissertation work, we developed new electroanalytical tools to probe chemical transformations/reactions at micro- and nano-interfaces and made use of new reaction schemes that capitalize on multiphase microenvironments.</p><p dir="ltr">Much of the method development was built on the foundation of stochastic nanoelectrochemistry, a technique that is reviewed thoroughly in Chapters 2, 4, and 5. Briefly, stochastic nanoelectrochemistry is the measurement of single nano-entities, one-at-a-time, as the collide with a micron-sized electrode. The nano-entities studied in this dissertation were aqueous droplets, either suspended in an immiscible oil continuous phase or propelled through air. We dove deeply into these studies, from using correlated microscopy to watch how these micro- and nanodroplets collide with other interfaces to building simulations to quantify changes to the chemistry inside. We showed how the surface environment directs water nanodroplet collisions (Chapter 10) and measured the sub-diffraction-limited nanometer contact area that forms between a microdroplet and a metal surface (Chapter 11). Using the nanodroplets as tiny reactors, we measured accelerated rate constants and promoted unfavorable nucleation events in attoliter-femtoliter aqueous droplets (see Chapter 6-7 and Chapter 12, respectively) and in microliter aqueous droplets (see Chapter 8 and Chapter 9, respectively).</p><p dir="ltr">As mentioned above, microdroplets are ubiquitous in air (<i>e.g.,</i> aerosols). However, electrochemistry is not an obvious choice for the measurement of intact aerosols because electrochemistry is traditionally performed in a conductive solution, and electrochemistry in air is difficult. In this dissertation we laid the groundwork for a path forward that allows electrochemical access the air|microdroplet interface. We designed and characterized a novel electrochemical cell, where the working electrode is a microwire traversing a suspended liquid film (Chapters 13-15). The early results were born from pure curiosity: Can we do electrochemistry in a soap bubble wall? Chapter 13 shows that the answer is “Yes!”, and that electrochemistry can report on aerosol contents that are collected from the air into this thin film. However, the soap bubble wall was severely limited by the lifetime of the bubble wall (bubbles pop), so in Chapters 14 and 15, we introduce a suspended ionic liquid film that does not pop from evaporation. With the more robust system, we realized the ability to probe intact single microdroplets, one-at-a-time (Chapter 14), giving electrochemical access to the air|water interface.</p><p dir="ltr">As detection of illicit substances from aerosols has the potential for immediate impact on first responder, user, and bystander safety, we employed the new technology to electroanalyze aerosolized methamphetamine (Chapter 13) and fentanyl (Chapter 15). Electrochemistry is small, simple, and affordable, making it a realistic candidate for an in-field sensor. We overcame selectivity challenges by using our understanding of interfacial microenvironments to leverage local pH changes, as demonstrated by the reliable detection of low purity cocaine in mixed powders (Chapter 16). This patented method provides a highly selective technique for cocaine identification in the presence of adulterants without the need to bring any chemicals to the scene (water is our only reagent!).</p><p dir="ltr">In sum, this body of work contributes to the electrochemical studies in nano- and microdroplets, extending the reach to account for droplet size on measured rates and to include microdroplets with a water|air boundary. Applications of the work were focused on in-field detection of illicit substances.</p>

<p>This thesis presents and uses the techniques of computational chemistry to explore two different processes induced in human skin by ultraviolet light. The first is the transformation of urocanic acid into a immunosuppressing agent, and the other is the enzymatic action of the 8-oxoguanine glycosylase enzyme. </p><p>The photochemistry of urocanic acid is investigated by time-dependent density functional theory. Vertical absorption spectra of the molecule in different forms and environments is assigned and candidate states for the photochemistry at different wavelengths are identified. </p><p>Molecular dynamics simulations of urocanic acid in gas phase and aqueous solution reveals considerable flexibility under experimental conditions, particularly for for the <i>cis</i> isomer where competition between intra- and inter-molecular interactions increases flexibility. </p><p>A model to explain the observed gas phase photochemistry of urocanic acid is developed and it is shown that a reinterpretation in terms of a mixture between isomers significantly enhances the agreement between theory and experiment , and resolves several peculiarities in the spectrum. </p><p>A model for the photochemistry in the aqueous phase of urocanic acid is then developed, in which two excited states governs the efficiency of photoisomerization. The point of entrance into a conical intersection seam is shown to explain the wavelength dependence of photoisomerization quantum yield. </p><p>Finally some mechanistic aspects of the DNA repair enzyme 8-oxoguanine glycosylase is investigated with density functional theory. It is found that the critical amino acid of the active site can provide catalytic power in several different manners, and that a recent proposal involving a S<i>N</i>1 type of mechanism seems the most efficient one.</p>

This thesis presents and uses the techniques of computational chemistry to explore two different processes induced in human skin by ultraviolet light. The first is the transformation of urocanic acid into a immunosuppressing agent, and the other is the enzymatic action of the 8-oxoguanine glycosylase enzyme. The photochemistry of urocanic acid is investigated by time-dependent density functional theory. Vertical absorption spectra of the molecule in different forms and environments is assigned and candidate states for the photochemistry at different wavelengths are identified. Molecular dynamics simulations of urocanic acid in gas phase and aqueous solution reveals considerable flexibility under experimental conditions, particularly for for the cis isomer where competition between intra- and inter-molecular interactions increases flexibility. A model to explain the observed gas phase photochemistry of urocanic acid is developed and it is shown that a reinterpretation in terms of a mixture between isomers significantly enhances the agreement between theory and experiment , and resolves several peculiarities in the spectrum. A model for the photochemistry in the aqueous phase of urocanic acid is then developed, in which two excited states governs the efficiency of photoisomerization. The point of entrance into a conical intersection seam is shown to explain the wavelength dependence of photoisomerization quantum yield. Finally some mechanistic aspects of the DNA repair enzyme 8-oxoguanine glycosylase is investigated with density functional theory. It is found that the critical amino acid of the active site can provide catalytic power in several different manners, and that a recent proposal involving a SN1 type of mechanism seems the most efficient one.

The reaction mechanisms for the hemoglobin degrading enzymes in the Plasmodium falciparum malaria parasite, plasmepsin II (Plm II) and histo-aspartic protease (HAP), have been analyzed by molecular simulations. The reaction free energy profiles, calculated by the empirical valence bond (EVB) method in combination with molecular dynamics (MD) and free energy perturbation (FEP) simulations are in good agreement with experimental data. Additional computational methods, such as homology modelling and automated substrate docking, were necessary to generate a 3D model and a reactive substrate conformation before the reaction mechanism in HAP could be investigated. HAP is found to be an aspartic protease with a peptide cleaving mechanism similar to plasmepsin II. The major difference between these enzymes is that the negatively charged tetrahedral intermediate is stabilized by the charged histidine in HAP while in Plm II it is a neutral aspartic acid. Also the reaction mechanism for two other aspartic proteases, cathepsin D and HIV-1 protease, was simulated. These enzymes are relevant both for the inhibitor selectivity and for obtaining a general picture of catalysis in aspartic proteases. Another project involves inhibitor design towards plasmepsins. In particular, Plm II directed inhibitors based on the dihydroxyethylene scaffold have been characterized computationally. Molecular dynamics (MD) simulations were used to propagate the investigated system through time and to generate ensembles used for the calculation of free energies. The ligand binding affinities were calculated with the linear interaction energy (LIE) method. The most potent inhibitor had a Ki value of 6 nM and showed 78 % parasite inhibition when tested on red blood cells infected by malaria parasite P. falciparum. Citrate synthase is part of the citric acid cycle and is present in organisms that live in cold sea water as well as hot springs. The temperature adaptation of citrate synthase to cold and heat was investigated in terms of the difference in transition state stabilization between the psychrophilic, mesophilic and hyperthermophilic homologues. The EVB, FEP and MD methods were used to generate reaction free energy profiles. The investigated energetics points toward the electrostatic stabilization during the reaction as the major difference between the different citrate synthase homologues. The electrostatic stabilization of the transition state is most effective in the following order of the citrate synthase homologues: hyperthermophile, mesophile, psycrophile. This could be a general rule for temperature adaptation of enzyme catalysis.

This  thesis  is  divided  into  four  parts,  all  centered  around  Constitutional Dynamic  Chemistry  (CDC)  and  Dynamic  Kinetic  Resolution  (DKR)  using biocatalysts for selective transformations, and their applications in screening of bioactive compounds, organic synthesis, and enzyme classification.    In  part  one,  an  introduction  to  CDC  and  DKR  is  presented,  illustrating  the basic  concepts,  practical  considerations  and  potential  applications  of  such dynamic systems, thus providing the background information for the studies in the following chapters.   In part two, Dynamic Systemic Resolution (DSR), a concept based on CDC is exemplified.  With  enzyme-catalyzed  transformations  as  external  selection pressure,  optimal  structures  can  be  selected  and  amplified  from  the  system. This  concept  is  expanded  to  various  types  of  dynamic  systems  containing single, double cascade/parallel, and multiple reversible reactions. In addition, the  substrate  selectivity  and  catalytic  promiscuity  of  target  enzymes  are  also investigated.   In   part   three,   DKR   protocols   using   reversible   reactions   for   substrate racemizations  are  illustrated.  Biocatalysts  are  here  employed  for  asymmetric transformations,  resulting  in  efficient  synthetic  pathways  for  enantioenriched organic compounds.   Part  four  demonstrates  two  unique  applications  of  CDC:  one  resulting  in enzyme  classification  by  use  of  pattern  recognition  methodology;  the  other involving  enzyme  self-inhibition  through  in  situ  transformation  of  stealth inhibitors employing the catalytic activity of the target enzyme. / <p>QC 20130614</p>