Spelling suggestions: "subject:"enzyme dynamics"" "subject:"anzyme dynamics""
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Substrate specificity and conformational activation mechanism of beta-phosphoglucomutaseSaltzberg, Daniel John 22 January 2016 (has links)
Phosphate transfer is ubiquitous in nature, however the occurance of phosphomutases is rare. Their uniqueness can be attributed to the complex and malleable substrate recognition scheme that allows the enzyme to perform two similar, yet distinct, catalytic steps while maintaining strict fidelity for substrate versus water. The complexity of developing this mechanism is highlighted in that, while phosphomutase function has independently evolved in most larger phosphotransferase superfamilies, very little diversification of this function has developed. As such, phosphomutases provide a rich framework to study the intricate specificity mechanisms employed by enzymes.
β-Phosphoglucomutase (bPGM) catalyzes the interconversion between β-glucose 1-phosphate (βG1P) and glucose 6-phosphate (G6P) via a β-glucose 1,6 bisphosphate (βG16P) intermediate. βPGM is in one of two subfamilies that have independently acquired phosphomutase activity within the ubiquitous Haloalkanoate Dehalogenase superfamily (HADSF) of phosphotransferases. The enzyme has been observed to undergo a large conformational change upon binding βG16P as well as a repositioning of the general acid/base catalyst residue Asp10. In addition, the mechanism involves cycling of the protonation state of Asp10, which requires a significant pKa shift. The importance of Asp10 and its activation of the enzyme have been discussed previously, however a clear understanding of the interplay between the conformational and catalytic activation mechanisms for βPGM has not been described.
This work uses aqueous phase techniques, solution X-ray scattering and molecular dynamics, to probe the effect of individual ligand moieties on the conformational state of the enzyme and free energy molecular dynamics and electrostatic calculations determine the interplay between conformation, protonation and Asp10 activation. The results implicate a model where the ligand-induced conformational change is governed by the non-catalytic phosphate site, and this transition induces correct positioning of Asp10, which, in turn induces the pKa shift, forming the catalytically competent complex.
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Novel Insights in Structure and Mechanism of Escherichia coli TransketolaseRabe von Pappenheim, Fabian 23 May 2017 (has links)
No description available.
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The Dynamics of Dehydrogenases - A Phase Space OdysseyVarga, Matthew J., Varga, Matthew J. January 2017 (has links)
Enzymes are immensely powerful and efficient heterogenous catalysts which are essential for life. As essential to life as enzymes are, it is still not well understood exactly how they enhance the rate of their catalyzed reactions up to 19 orders of magnitude over their solution phase counterpart reactions. Recent research has focused on sub--picosecond motions coupled to the reaction coordinate, called rate--promoting vibrations, which are important components of several well--known enzymatic mechanisms and build upon previous models of enzyme activity. Herein I present two studies which are expressly focused on providing tools and knowledge to understand how dynamics affects enzymatic reactions. First, I present a method for the calculation of kinetic isotope effects from first principles, using transition path sampling and centroid molecular dynamics. This method allows for the calculation of kinetic isotope effects without the assumptions necessitated by transition state theory or free energy perturbation methods. It was found that this method could calculate the primary H/D kinetic isotope effect of the conversion of benzyl alcohol to benzaldehyde in yeast alcohol dehydrogenase to within the margin of error of experimentally measured kinetic isotope effects of the same reaction. Second, I examined the role that evolution plays in the preservation of these rate--promoting vibrations, by performing a transition path sampling study of two lactate dehydrogenases, those of Plasmodium falciparum and Cryptosporidium parvum, which evolved through separate gene duplication events from a common malate dehydrogenase ancestor. It was found that though both lactate dehydrogenases share the same rate--promoting vibration, and indeed share the rate--promoting vibration found in other lactate dehydrogenases, the sequence variations in lactate dehydrogenase from P. falciparum causes a diminished contribution of the motions to the reaction coordinate. The studies presented in this dissertation contribute to the our understanding of enzymes on an atomistic level, as well as providing tools necessary for designing novel de novo enzymes and targeted drugs for enzymes of disease--causing organisms.
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Ultrafast Dynamics of Intramolecular Electron Transfer and DNA Repair by PhotolyaseLiu, Zheyun 04 September 2013 (has links)
No description available.
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Using Molecular Dynamics to Elucidate the Mechanism of CyclophilinMcGowan, Lauren 09 May 2014 (has links)
Cyclophilins are ubiquitous enzymes that are involved in protein folding, signal transduction, viral proliferation, oncogenesis, and regulation of the immune system. Cyclophilin A is the prototype of the cyclophilin family. We use molecular dynamics to describe the catalytic mechanism of cyclophilin A in full atomistic detail by sampling critical points along the reaction coordinate, and use accelerated molecular dynamics to sample cis-trans interconversions. At these critical points, we analyze the conformational space sampled by the active site, flexibility of the enzyme backbone, and modulation of binding interactions.We use Kramer’s rate theory to determine how diffusion and free energy contribute to lowering the activation energy of prolyl isomerization. We also find preferential binding modes of several cyclophiln A inhibitors, and compare the conformational space sampled by inhibited cyclophilin A to the conformational space sampled during wild-type interactions. We also analyze the mechanism of the next family member cyclophilin B in order to probe differences in enzyme dynamics and intermolecular interactions that could possibly be exploited in isoform-specific drug design. Our results indicate that cyclophilin proceeds by a conformational selection binding mechanism that manipulates substrate sterics, electrostatic interactions, and multiple reaction timescales in order to speed up reaction rate. Conformational space sampled by cyclophilin when inhibited and when undergoing wild-type interactions share significant similarity. Cyclophilins A and B do have notable differences in enzyme dynamics, due to variation in intramolecular interactions that arise from variation in primary structures. This work demonstrates how computational methods can be used to clarify catalytic mechanisms.
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NITROREDUCTASE: EVIDENCE FOR A FLUXIONAL LOW-TEMPERATURE STATE AND ITS POSSIBLE ROLE IN ENZYME ACTIVITYZhang, Peng 01 January 2007 (has links)
The enzyme nitroreductase (NR) catalyzes two-electron reduction of high explosives such as trinitrotoluene (TNT), a wide variety of other toxic nitroaromatic compounds, as well as riboflavin derivatives, using a tightly-bound flavin mononucleotide (FMN) cofactor. It has important environmental and clinical implications. Previous studies have focused on elucidating NRs catalytic mechanism and solving its crystal structure.
In this dissertation work, we first develop and implement new strategies for labeling NR with stable isotopes, and report a completely re-designed protocol for NRs purification. Then we report the successful assignment of over half of NRs backbone resonances using 3d-NMR methods. The most significant observation is that we find a well-resolved 2d 1H-15N HSQC NMR spectrum is obtained at 37°C for NR, while the HSQC at 4°C is poorly-dispersed and comprised of sharp overlapped peaks. Thus, it would appear that NR denatures at 4°C. However, as we demonstrate, the non-covalently-bound FMN cofactor is not released at the lower temperature, based on retention of the native flavin visible-CD spectrum. Similarly, far-UV CD spectroscopy shows that the protein retains full secondary structural content at 4C. In addition, near-UV CD and Fluorine-19 NMR experiments demonstrate that under completely native conditions (neutral pH, no additives) NR maintains a high degree of tertiary structure and well-defined hydrophobic side-chain packing, ruling out the possibility of a molten-globule state.
Thus, our studies report strong evidence that the dramatic low-temperature (low-T) transition near 20°C observed by NMR is not the result of protein structural changes, but rather, we propose that NR exists as an ensemble of rapidly inter-converting structures, at lower temperature, only adopting a well-defined unique structure above 20°C. Both saturation-transfer from water and solvent proton-exchange measurements support our proposed model in which the unique high-T structure is favored entropically, by release of water molecules; on the other hand, the fluxional low-T state incorporates greater water solvation at 4°C.
In the latter part of this study, we present preliminary data suggesting that the flexibility implied by easy water-access to the loosely-structured state plays a role in accommodating binding of diverse substrates. Therefore, the fluxional low-T state may be functionally important. A possible direct link between the enzyme dynamics and its catalytic activity will be an area of future investigation.
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The use of kinetic isotope effects in studies of hydrogen transfersRoston, Daniel Harris 01 December 2013 (has links)
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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Computational Perspective on Intricacies of Interactions, Enzyme Dynamics and Solvent Effects in the Catalytic Action of Cyclophilin ATork Ladani, Safieh 11 May 2015 (has links)
Cyclophilin A (CypA) is the well-studied member of a group of ubiquitous and evolutionarily conserved families of enzymes called peptidyl–prolyl isomerases (PPIases). These enzymes catalyze the cis-trans isomerization of peptidyl-prolyl bond in many proteins. The distinctive functional path triggered by each isomeric state of peptidyl-prolyl bond renders PPIase-catalyzed isomerization a molecular switching mechanism to be used on physiological demand. PPIase activity has been implicated in protein folding, signal transduction, and ion channel gating as well as pathological condition such as cancer, Alzheimer’s, and microbial infections.
The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerization by CypA has been the target of intense research. Normal and accelerated molecular dynamic simulations were carried out to understand the catalytic mechanism of CypA in atomistic details. The results reaffirm transition state stabilization as the main factor in the astonishing enhancement in isomerization rate by enzyme. The ensuing intramolecular polarization, as a result of the loss of pseudo double bond character of the peptide bond at the transition state, was shown to contribute only about −1.0 kcal/mol to stabilizing the transition state. This relatively small contribution demonstrates that routinely used fixed charge classical force fields can reasonably describe these types of biological systems. The computational studies also revealed that the undemanding exchange of the free substrate between β- and α-helical regions is lost in the active site of the enzyme, where it is mainly in the β-region. The resultant relative change in conformational entropy favorably contributes to the free energy of stabilizing the transition state by CypA. The isomerization kinetics is strongly coupled to the enzyme motions while the chemical step and enzyme–substrate dynamics are in turn buckled to solvent fluctuations. The chemical step in the active site of the enzyme is therefore not separated from the fluctuations in the solvent. Of special interest is the nature of catalysis in a more realistic crowded environment, for example, the cell. Enzyme motions in such complicated medium are subjected to different viscosities and hydrodynamic properties, which could have implications for allosteric regulation and function.
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Aldolases for Enzymatic Carboligation : Directed Evolution and Enzyme Structure-Function Relationship StudiesMa, Huan January 2015 (has links)
The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6-phosphate aldolase (FSA). Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attention as environmentally friendly alternative for the synthesis of polyhydroxylated compounds in stereocontrolled manner. However, naturally occurring aldolases can hardly be used directly in organic synthesis mainly due to their narrow substrate scopes, especially phosphate dependency on substrate level. Semi-rational directed evolution was used in order to investigate the possibility of expanding the substrate scope of both DERA and FSA and to understand more about the relationship between protein structure and catalytic properties. The first two projects focus on the directed evolution of DERA and studies of the enzyme mechanism. The directed evolution project aims to alter the acceptor substrate preference from phosphorylated aldehydes to aryl-substituted aldehydes. Effort has been made to develop screening methods and screen for variants with desired properties. In the study of enzyme mechanism where enzyme steady state kinetic studies were combined with molecular dynamic simulations, we investigated the role of Ser238 and Ser239 in the phosphate binding site and the possible connection between enzyme dynamics and catalytic properties. The other two projects focus on the directed evolution of FSA and the development of a new screening assay facilitating screening for FSA variants with improved activity in catalyzing aldol reaction between phenylacetaldehyde and hydroxyacetone. The new assay is based on a coupled enzyme system using an engineered alcohol dehydrogenase, FucO DA1472, as reporting enzyme. The assay has been successfully used to identify a hit with 9-fold improvement in catalytic efficiency and to determine the steady state kinetic parameters of wild-type FSA as well as the mutants. The results from directed evolution illustrated the high degree malleability of FSA active site. This opens up possibilities to generate FSA variants which could utilize both aryl-substituted donor and acceptor substrates.
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Investigating the contribution of protein dynamics to catalysis in protochlorophyllide oxidoreductaseHoeven, Robin January 2015 (has links)
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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