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Ligand Interactions at the Active Site of Aspartate Transcarbamoylase from Escherichia ColiDennis, Paul 03 1900 (has links)
The carbamoyl region of the active site of aspartate transcarbamoylase from Escherichia coli was probed using an enzyme assay in which the two substrates were varied near their respective K_m 's. The inhibitors tested, some synthesized and some commercially available, were chosen to satisfy the structural requirements for binding to either the dicarboxylate or phosphate region with a substituent capable of extending into the carbamoyl region. However, the dicarboxylate based inhibitors were found to bind in an abnormal manner (unlike L-aspartate or succinate on which they were based). The carbamoyl region was found to contain a positively charged side-chain and preliminary results indicate that tetrahedral groups are not preferred over trigonal moieties. It is suggested that electrostatic stabilization of the negative charge which develops in the transition state may be a major factor in promoting catalysis. The identity of this charged group in the carbamoyl region is postulated to be His134 based on available X-ray diffraction data. The binding subsites of the active site of this enzyme were also found to be oriented in essentially the same plane. These results will greatly aid in the design of future mechanism-based
inhibitors to this enzyme that may have therapeutic value (at this time the mammalian enzyme is thought to have a similar catalytic mechanism). / Thesis / Master of Science (MS)
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INDUCING ACTIVE SITES IN CLUSTERS: REACTIVITY OF Al13Ix- and Al14Iy- (x=0-2, y=2-4) WITH METHANOLPowell, Christopher 06 May 2011 (has links)
Size selective reactivity has been observed in pure aluminum cluster anions as a result of Lewis acid and base pairs. Using this a starting point, the goal of this study has been to explore how reactivity is affected with the addition of one or more ligand, which may induce active sites on the surface of the metal clusters. To study this, a theoretical investigation was undertaken on Al13Ix- and Al14Iy- (x=0-2, y=2-4) and their reactivity with methanol. The hypothesis was that iodine can induce a Lewis base site on the opposite side of the cluster, which may enhance reactivity. In results that are consistent with preliminary experimental data, it was found that the Al13Ix- series has a large energy barrier with respect to the cleavage of the O-H bond of methanol. The clusters of the series act as an extremely poor Lewis acids, and as a result, these clusters are relatively inert to methanol etching. On the other hand, the Al14Iy- series has a low barrier and is expected to react rapidly with methanol. The series is found to be most reactive at an aluminum adatom that is bound to an iodine due to the iodine extracting charge from the aluminum cluster creating a strong Lewis acid site.
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On the Mechanistic Roles of the Protein Positive Charge Close to the N(1)Flavin Locus in Choline OxidaseGhanem, Mahmoud 12 June 2006 (has links)
Choline oxidase catalyzes the oxidation of choline to glycine betaine. This reaction is of considerable medical and biotechnological applications, because the accumulation of glycine betaine in the cytoplasm of many plants and human pathogens enables them to counteract hyperosmotic environments. In this respect, the study of choline oxidase has potential for the development of a therapeutic agent that can specifically inhibit the formation of glycine betaine, and therefore render pathogens more susceptible to conventional treatment. The study of choline oxidase has also potential for the improvement of the stress resistance of plant by introducing an efficient biosynthetic pathway for glycine betaine in genetically engineered economically relevant crop plant. In this study, codA gene encoding for choline oxidase was cloned. The cloned gene was then used to express and purify the wild-type enzyme as well as to prepare selected mutant forms of choline oxidase. In all cases, the resulting enzymes were purified to high levels, allowing for detailed characterizations. The biophysical and biochemical analyses of choline oxidase variants in which the positively charged residue close to the flavin N(1) locus (His466) was removed (H466A) or reversed (H466D) suggest that in choline oxidase, His466 modulates the electrophilicity of the bound flavin and the polarity of the active site, and contributes to the flavinylation process of the covalently bound FAD as well as to the stabilization of the negative charges in the active site. Biochemical, structural, and mechanistic relevant properties of selected flavoproteins with special attention to flavoprotein oxidases, as well as the biotechnological and medical relevance of choline oxidase, are presented in Chapter I. Chapter II summarizes all the experimental techniques used in this study. Chapter III-VII illustrate my studies on choline oxidase, including cloning, expression, purification and preliminary characterizations (Chapter III), spectroscopic and steady state kinetics (Chapter IV), the catalytic roles of His466 and the effects of reversing the protein positive charge close to the flavin N(1) locus (Chapter V and VI), and the roles of His310 with a special attention to its involvement in a proton-transfer network (Chapter VII). Chapter VIII presents a general discussion of the data presented.
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The Calpain Protease Active Site: A Target for Inhibitor and Activity-Based Probe DesignQian, Jin 04 September 2008 (has links)
The calpain family of intracellular Ca2+-dependent cysteine proteases is involved in a number of intracellular signaling processes. Calpain hyperactivity has also been implicated in ischemic injury, neurodegenerative diseases and cataract formation. However, the specific function of calpains in these normal and diseased states remains unclear. Competitive inhibition of calpain is useful for studying their functions and can lead to pharmacological treatments, while monitoring their activity with activity-based probes (ABPs) can reveal how calpain is regulated and be applied to screen for inhibitors in vivo. But these strategies are complicated by the similarity of the calpain active-site when compared to other intracellular cysteine proteases. Therefore, there is a need to design inhibitors and ABPs that selectively target calpain. Using X-ray crystallography, the interactions between the calpain active-site and each of two reversible inhibitors was studied. This led to the discovery of novel non-covalent aromatic stacking and hydrogen bonding interactions between the primed-side adenine group of one inhibitor and indole ring of an active-site Trp residue in μ-calpain. A substrate-based competition assay later confirmed that these interactions provided this compound with an inhibitory advantage over the other, which lacked any primed-side interactions, thereby providing insight into the development of new, more specific reversible calpain inhibitors. Next, a fluorescent ABP, containing features borrowed from an irreversible and presumably calpain-specific inhibitor, was evaluated for its ability to detect calpain activitiy. Although this probe appropriately targeted the calpain active site in its Ca2+-activated form, it was unable to detect calpain activity in a cell extract. Nevertheless, the results of this study have yielded insights into ways of improving the calpain detecting ability of this ABP. / Thesis (Master, Biochemistry) -- Queen's University, 2008-09-01 15:39:07.023
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Atomistic Insights into Binding Pocket Dynamics and Regulation in the Interleukin-2 T-Cell Kinase SH2 DomainMomin, Mohamed 08 August 2017 (has links)
Although the regulation of proteins functions by allosteric interactions has been identified in many subcellular processes, long-range conformational changes in proteins are also known to be induced by molecular switches. A molecular switch based on the cis-trans isomerization of a peptidyl-prolyl bond is capable of inducing a conformational change directly to the protein backbone, which is then propagated throughout the system. However, these switches are elusive and difficult to identify due to their intrinsic dynamics in the biomolecules where they are found. Herein, we explore the conformational dynamics and free energy landscape of the SH2 domain of Interleukin-2-inducible T-Cell Kinase (ITK) to fully understand the conformational coupling between the distal cis-trans molecular switch, and its phosphotyrosine binding pocket. Using multiple microsecond-long all-atom molecular dynamics simulations in explicit water for over a total of 60 μs, we show that the cis-trans isomerization of the Asn286-Pro287 peptidyl-prolyl bond is directly correlated to the dynamics of the phosphotyrosine binding pocket, in agreement with previous NMR studies. While the cis state is localized to a single free energy basin and less dynamic, the trans state samples two distinct conformations of its binding pocket – one that recognizes the phosphotyrosine motif, and another that is similar the cis state. These results provide an atomic-level description of a less-well understood allosteric regulation by a peptidyl-prolyl cis-trans molecular switch that could aid in the understanding of normal and aberrant sub-cellular process and the identification of these elusive molecular switches in other proteins.
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Separation and Characterization of Variant Forms of Phosphoglucose Isomerase: Purification and Structural Analysis of Active Site Peptides from Human and Rabbit Phosphoglucose IsomeraseGibson, David R. 05 1900 (has links)
A method has been developed for the rapid, quantitative separation of normal and abnormal phosphoglucose isoemrase allozymes from individuals heterozygous for genetic variant forms of the enzyme. The method utilizes a substrate gradient elution of the enzyme from carboxymethyl Biogel and is far superior in terms of resolution and recovery to methods based on electrophoresis and isoelectric focusing. Four different genetic variant forms of the enzyme were isolated and subjected to a systematic comparison of their physical, catalytic and stability properties. The physical and catalytic properties of the variants were similar; however, clear differences in the stability of the allozymes were apparent.
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Molecular Dynamics Simulations of Pseudomonas cepacia lipase in aqueous solutionsVariyath, Samrish 20 April 2011 (has links)
No description available.
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ATP hydrolysis in Rho: Identifying active site residues and their rolesBalasubramanian, Krithika January 2010 (has links)
Escherichia coli transcription termination factor Rho is a hexameric RNA/DNA helicase that terminates transcription using energy derived from the hydrolysis of ATP. The ATP binding sites of Rho are located at the interfaces of adjoining subunit Cterminal domains and have the Walker A and B motifs, characteristic of many ATPases (Skordalakes & Berger, 2003; Richardson 2002). Available Rho crystal structures capture the protein with its active site in an open configuration that must close to permit ATP hydrolysis. Because of this, the identities of active site residues predicted to mediate ATP hydrolysis are uncertain. To determine which amino acids activate water, stabilize transition state, sense the γ- phosphoryl group, and coordinate the magnesium ion of MgATP, we have carried out site-specific mutagenesis on candidate residues which are conserved across bacterial species, and characterized the relevant properties of the mutant proteins. The residues chosen were E211 as the water activator, R212 as the γ sensor, R366 as the arginine finger, and D265 as the residue that coordinates Mg2+. Each mutant protein was investigated for its ability to oligomerize as hexamers, assayed for ATPase activity, ATP and RNA binding, and pre-steady-state kinetics. The results show that the mutant proteins form hexamers similarly as to wild type Rho. The RhoE211 mutants display at least a 200-fold lower activity as ATPases, bind both ATP and RNA with similar affinities as the wild type protein, and display no burst in pre-steady-state kinetics. RhoR212A protein has 20-fold lower activity as an ATPase compared to wild type Rho, binds ATP with at least a 50-fold weaker affinity, and RNA with a 2-fold higher KD compared to wild type Rho. RhoR366A functions as an ATPase with 50-fold lower activity, binds RNA with similar affinity as wild type Rho and binds ATP with a 5- fold weaker affinity. RhoD265N displays 150-fold lower ATPase activity compared to the wild type enzyme, binds ATP with a 10-fold weaker affinity, and binds RNA with similar affinity as wild type Rho. Pre-steady-state kinetics studies indicate that the mutant proteins investigated show no burst kinetics, indicating a failure or a significantly slower rate of the hydrolysis (chemistry) step. It is possible that the rate-limiting step is the chemistry step in these mutant proteins, contrary to the wild type protein where the chemistry step is much faster (300/s). Together, the results obtained are consistent with the proposed roles for these residues: E211 is involved in activating a water molecule, R212 functions as the γ sensor, R366 functions as the arginine finger and D265 is involved in coordination of the Mg2+ ion. This study has elucidated the mechanism of ATP hydrolysis, by determining some of the key residues involved in the hydrolysis reaction. This study is only a part of the characterization of the active site residues. There might be other residues involved in one or all of the functions proposed. Utilizing the findings from this study, other experiments and models can be implemented to understand how Rho hydrolyzes ATP and utilizes the energy to move along the RNA molecule and functions as a helicase. / Biochemistry
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Structure-Function Studies on Two Phosphoenolpyruvate CarboxylasesDharmarajan, Lakshmi 29 April 2011 (has links)
Phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (Pepc) are two important CO₂-fixation enzymes which share a similar reaction mechanism. Both operate through a lid-gated active site and have a hypothesized enol-pyruvate intermediate in their catalytic pathway. While PEPCK is an important metabolic enzyme in animals and plays a broad role in cataplerosis, gluconeogenesis and glyceroneogenesis, Pepc reaction in plants catalyzes the first committed step in CO₂ fixation in CAM and C₄ plants via Rubisco. We are studying the structure-function aspects of both enzymes, with a goal of discovering new elements in these enzymes which can modulate catalysis. We have undertaken an interdisciplinary approach for this work and have shown that a combination of experimental and computational techniques can be complementary and can provide novel information.
We have determined that in human PEPCK, Tyr235 forms an anion-quadrupole interaction with the carboxylate of PEP and thus positions the latter with respect to the enzyme-bound Mn²+ for optimal phosphoryl transfer and catalysis. We have also identified Pro82 as a catalytically influential residue in this enzyme. Using molecular dynamics simulations we have noted that absence of ligands induces active-site lid opening in GTP-PEPCKS and we have made the first observation of the intermediary structures of the lid opening event, the dynamics of which is an important element that controls GTP-PEPCK catalysis.
We have determined the first three-dimensional crystal structure of an archaeal-type Pepc, i.e. C. perfringens PepcA. Our experimental data also provide information about the oligomerization of PepcAs and reveal that aspartate inhibits the C. perfringens enzyme competitively compared to the allosteric inhibition in Pepcs. Structure-based modeling has led to the identification of putative aspartate- and bicarbonate-binding residues in C. perfringens PepcA, of which Arg82, His11, Ser201, Arg390, Lys340, Arg342 and Arg344 probably play an important role. / Ph. D.
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Molecular, Biochemical, and Toxicological Evaluation of Anticholinesterases for control of the Malaria Mosquito, Anopheles gambiaeMutunga, James Mutuku 26 May 2011 (has links)
Pyrethroids are the only class of insecticides approved by the World Health Organization (WHO) for use in insecticide treated nets (ITNs), the first line of malaria vector control. Widespread resistance development to pyrethroids undermines current control efforts, and hence an urgent need for alternative chemistries. I report the evaluation of pharmacological differences between insect and vertebrate acetylcholinesterase (AChE) as well as selectivity and toxicity testing of new carbamate insecticides on Anopheles gambiae, the African malaria mosquito. AChE gorge pharmacology data revealed differences between insect and vertebrate AChE that can be exploited in the design of a bivalent insecticide. Toxicokinetic analysis showed that metabolic detoxication and cuticular penetration affect toxicity of carbamates in a manner dependent on the chemical structure.
Structure activity relationships of side-chain branched N-methylcarbamates emphasized the importance of structural complementarity of ligands to the AChE catalytic active site and the substrate, acetylcholine. Monovalent pyrazoles and acetophenone oxime carbamates were toxic to both susceptible and carbamate-resistant mosquitoes carrying a G119S mutation within the catalytic site. A bivalent phthalimide-pyrazole carbamate and sulfenylated phenyl N-methyl carbamates were highly toxic when topically applied onto insect but less toxic by treated filter paper assays. In vitro evaluation of a molecular mosquito-selectivity model using AChE peripheral site ligands confirmed that selectivity of PRC 472 was due to presence of I70 in mosquito, which is Y70 in human AChE. The findings presented here are important steps in the on-going search of a mosquito-selective and resistance mitigating carbamate insecticide for control of malaria mosquitoes. / Ph. D.
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