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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

Studies of structure, function and mechanism in pyrimidine nucleotide biosynthesis

Harris, Katharine Morse January 2012 (has links)
Thesis advisor: Evan R. Kantrowitz / Thesis advisor: Mary F. Roberts / Living organisms depend on enzymes for the synthesis using small molecule precursors of cellular building blocks. For example, the amino acid aspartate is synthesized in one step by the amination of oxaloacetate, an intermediate compound produced in the citric acid cycle, exclusively by means of an aminotransferase enzyme. Therefore, function of this aminotransferase is critical to produce the amino acid. In the Kantrowitz Lab, we seek to understand the molecular rational for the function of enzymes that control rates for the biosynthesis of cellular building blocks. If one imagines the above aspartate-synthesis example as a single running conveyer belt, any oxaloacetate that finds its way onto that belt will be chemically transformed to give aspartate. We can extend this notion of a conveyer belt to any enzyme. Therefore, the rate at which the belt moves dictates the rate of synthesis. Now imagine many, many conveyer belts lined in a row to give analogy to a biosynthesis pathway requiring more than one enzyme for complete chemical synthesis. This is such the case for the biosynthesis of nucleotides and glucose. Nature has developed clever tricks to exquisitely control the rate of product output but means of altering the rate of one or some of the belts in the line of many, without affecting the rate of others. This type of biosynthetic rate regulation is termed allostery. Studies described in this dissertation will address questions of allosteric processes and the chemistry performed by two entirely different enzymes and biosynthetic pathways. The first enzyme of interest is fructose-1,6-bisphosphatase (FBPase) and its role in the biosynthesis of glucose. Following FBPase introduction in Chapter One, Chapter Two describes the minimal atomic scaffold necessary in a new class of allosteric type 2 diabetes drug molecules to effect catalytic inhibition of <italic>Homo sapiens</italic> FBPase. Following, is the second enzyme of interest, aspartate transcarbamoylase (ATCase) and its role in the biosynthesis of pyrimidine nucleotides. Succeeding ATCase introduction in Chapter Three, Chapter Four describes a body of work exclusively about the catalysis by ATCase. This work was inspired by the human form of the enzyme following the human genome project completion providing data that show likely <italic>Homo sapiens</italic> ATCase is not allosterically regulated. Chapter Five describes work on a allosterically-regulated, mutant ATCase and provides a biochemical model for the molecular rational for the catalytic inhibition upon cytidine triphosphate (CTP) binding to the allosteric site. The experimental techniques used for answering research questions were enzyme X-ray crystallography, <italic>in silico</italic> docking, kinetic assay experiments, genetic sub-cloning and genetic mutation. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Structural studies of giardial arginine deiminase

Suharto, Adrian Rinaldi, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2006 (has links)
Recombinant giardial arginine deiminase (rADI) was characterized. The enzyme was found to have a specific activity of 12 U (mg protein)-1under at pH 7.4 and 1 mM arginine. The maximum velocity was 14.75 U (mg protein-1) and the KM was 0.167 mM. The specific activity and maximum velocity values are significantly lower than the values reported previously for giardial rADI, while the KM value is quite similar. The optimum pH for the giardial rADI was 6-9, a broad range compared to other arginine deiminases. Recombinant ADI is very specific in its binding specificity, with canavanine (KI 2.4 mM) and ornithine (KI 2.1 mM) being the only substrate analogues giving significant inhibition from the wide variety of analogues tested. None of the analogues could be shown to act as alternative substrates. The contribution of conserved, catalytic and C-terminal residues proposed by previous research towards ADI activity was investigated by site-directed mutagenesis. Mutations of catalytic site residues Asp175, Glu226, His280 and Cys424 decreased the rADI activity to below 2%. Conservative mutations showed significant activity for Asp175 to Glu175 (DE175) and Glu226 to Asp226 (ED226). Site directed mutagenesis on the conserved non-catalytic site Leu46 showed activities below 15%, with the highest activity observed for the mutation to Val46 (LV46), which differs in one CH2 to Leu46 on its side chain. The KM of the mutant LV46 was 3.64 mM while for LA46 (Leu to Ala mutation) was 1.33 mM. Excising 5, 13, 16 amino acids from the C-terminal residues resulted in activity decreasing to 0.8% of the wild type, while excising 54 amino acids caused the rADI to be insoluble. Sequence alignment of Giardia and Dictyostelium suggests a homologous area within the C-terminal extension. Site directed mutagenesis on the Tyr567 residue in this region resulted in a decrease in activity, with the highest activity observed for a Tyr to Phe mutation. Studies using specific cysteine protease inhibitors demonstrated partial protection against proteolysis of ADI against giardial proteases. Degradation of ADI by giardial proteases was more rapid at pH 6 than at pH 7.4.

Separationless immunoassay and DNA sensing using wired enzyme based amperometric affinity electrodes /

Campbell, Charles Nelson, January 2000 (has links)
Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 220-243). Available also in a digital version from Dissertation Abstracts.

An efficient assay for identification and quantitative evaluation of potential polysialyltransferase inhibitors

Guo, Xiaoxiao, Malcolm, Jodie R., Ali, Marrwa M., Ribeiro Morais, Goreti, Shnyder, Steven, Loadman, Paul, Patterson, Laurence H., Falconer, Robert A. 08 May 2020 (has links)
Yes / The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors. / Yorkshire Cancer Research, Wellcome Trust

Production and properties of a protease secreted by Pseudomonas fluorescens R8

Zahran, Ahmed Shawky January 1988 (has links)
No description available.

Exploring the structure and function of bacterial cytosine specific DNA methyltransferases using site-directed mutagenesis

Sheikh, Qaiser Iftikhar January 2001 (has links)
Point mutations were engineered into the sequence of the multispecific DNA methyltransferase (Mtase) M. SPRI in motif IX, in order to mimic the corresponding motif IX of mono-specific Mtase. A similar approach was adopted to modify the sequence of the monospecific enzyme M. HhaI in motifs IX and X based on the available structure and as a consequence the enzyme regained methylation potential. It was thought that these changes might be sufficient to enable functional exchange of the target recognition domains (TRDs) between a mono- and a multispecific enzyme. However, insertion of various segments of TRD region from M. SPRI into the M. HhaI was not successful (Chapter 4). To establish whether mono- and multispecific Mtases are incompatible in terms of sequence exchanges, a systematic "swapping" of motifs was carried out (Chapter 5). These experiments suggested that there are some enzyme-specific structural interactions between different subunits within each class of Mtases. In second half of this thesis a bacterial two-hybrid system based on the reversible assembly of an engineered form of M. SPRI was developed (Chapter 6). However the Mtase protein does not assemble into an active species until a DNA segment encoding a leucine zipper motif is fused to each of the two halves. Co-transformation of E. coli with the plasmids expressing the C-terminal and N-terminal domains respectively resulted in the abolition of colonies on double antibiotic plates, when an mcr strain was used as host. High performance liquid chromatography was used to estimate the extent of modification of plasmids indirectly. The extent of methylation at specific sequences within a plasmid molecule was readily detected by the corresponding differential susceptibility to digestion by specific restriction enzymes. Using this approach it proved possible to detect different levels of activity produced by wild type and mutant recombinant DNA Methyltransferases with sensitivity and in a semi quantitative manner. In order to analyse the biochemical properties of Mtase, I have developed an in vitro translation-modification assay. Binary studies with the mutants (from Chapter 3 and 5) showed that there were no detectable sequence-specific recognition differences between these enzymes. Taken together, these results suggest that motif IX plays a role in general stabilisation of the enzyme core structure and has a less significant role in DNA recognition.

Studies of enzyme induction in rape (Brassica napus) seeds

Broadhurst, T. P. January 1986 (has links)
No description available.

Mammalian 2-oxo acid dehydrogenase multienzyme complexes and the dihydrolipoamide dehydrogenase component

Wawrzynczak, E. J. January 1984 (has links)
No description available.

Studies on tRNA charging by tyrosyl-tRNA synthetase

Avis, Johanna M. January 1991 (has links)
No description available.

The role of phospholipases, MAPK's and cyclooxygenases in tumour growth

Mckay, Diane Tracey January 2001 (has links)
No description available.

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