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Characterization of Aspartate Transcarbamoylase in the Archaebacterium Methanococcus JannaschiiStewart, John E. B. (John Edward Bakos) 12 1900 (has links)
Asparate transcarbamoylase catalyzes the first committed step in the de novo synthesis of pyrmidine nucleotides UMP, UDP, UTP, and CTP. The archetype enzyme found in Escherichia coli (310 kDa) exhibits sigmodial substrate binding kinetics with positive control by ATP and negative control with CTP and UTP. The ATCase characterized in this study is from the extreme thermophilic Archaebacterium, Methanococcus jannaschii. The enzyme was very stable at elevated temperatures and possessed activity from 20 degrees Celsius to 90 degrees Celsius. M. Jannaschii ATCase retained 75% of its activity after incubation at 100 degrees Celsius for a period of 90 minutes. No sigmodial allosteric response to substrate for the enzyme was observed. Velocity substrate plots gave Michaelis-Menten (hyperbolic) kinetics. The Km for aspartate was 7 mM at 30 degrees Celsius and the KM for carbamoylphosphate was .125 mM. The enzyme from M. jannaschii had a broad pH response with an optimum above pH 9. Kinetic measurements were significantly affected by changes in pH and temperature. The enzyme catalyzed reaction had an energy of activation of 10,300 calories per mole. ATCase from M. jannaschii was partially purified. The enzyme was shown to have a molecular weight of 110,000 Da., with a subunit molecular weight of 37,000 Da. The enzyme was thus a trimer composed of three identical subunits. The enzyme did not possess any regulatory response and no evidence for a regulatory polypeptide was found, DNA from M. jannaschii did hybridize to probes corresponding to genes for both the catalytic and regulatory subunits from E. coli. Analysis of DNA sequences for the M. jannaschii ATCase genes showed that the gene for the catalytic subunits shares significant homology with the pyrB genes from E. coli, and maximum homology amongst known ATCase genes to pyrB from Bacillus. An unlinked gene homologous to E. coli pyrl encoding the regulatory subunit was identified, though its expression and true function remain uncharacterized.
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Isolation of a Pseudomonas aeruginosa Aspartate Transcarbamoylase Mutant and the Investigation of Its Growth Characteristics, Pyrimidine Biosynthetic Enzyme Activities, and Virulence Factor ProductionHammerstein, Heidi Carol 12 1900 (has links)
The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.
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Isolation and Characterization of the Operon Containing Aspartate Transcarbamoylase and Dihydroorotase from Pseudomonas aeruginosaVickrey, John F. (John Fredrick), 1959- 05 1900 (has links)
The Pseudomonas aeruginosa ATCase was cloned and sequenced to determine the correct size, subunit composition and architecture of this pivotal enzyme in pyrimidine biosynthesis. During the course of this work, it was determined that the ATCase of Pseudomonas was not 360,000 Da but rather present in a complex of 484,000 Da consisting of two different polypeptides (36,000 Da and 44,000 Da) with an architecture similar to that of E. coli ATCase, 2(C3):3(r2). However, there was no regulatory polypeptide found in the Pseudomonas ATCase.
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EPR spin-trapping studies of radical reactions with DNA basesHo, Win Fung January 1998 (has links)
No description available.
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Studies of the chemistry of some 5-diazopyrimidine nucleosidesYin, Zhong January 1994 (has links)
No description available.
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Synthesis of pyrimidine C-nucleoside analogues and triphosphate derivativesChan, Heng Ming January 2008 (has links)
Five pyrimidine C-nucleosides were prepared via Heck-type coupling reactions. These derivatives are designed to mimic dC and dU (or T). The minor groove O2 carbonyl in each derivative is replaced by a hydrogen, a fluorine, or a methyl group. The hydrogen-substituted dC analogue was converted into a 2’,3’-dideoxynucleoside, which was converted into a 5’-triphosphate derivative. The other two dC analogues were transformed into 5’-triphosphate derivatives immediately after Heck coupling reactions. These analogues will allow an examination of the nature and role of minor groove interactions between incoming triphosphates and various polymerases. / Thesis (MS) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
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The effects of protein associations on pyrimidine deoxyribonucleotide biosynthesisMcGaughey, Kathleen M. 29 November 2001 (has links)
The faithful replication of DNA depends on the appropriate balance of DNA
precursors. From studies conducted in bacteriophage T4, models for
deoxyribonucleotide biosynthesis producing pools appropriate for DNA replication
have made it possible to understand more complex systems. A portion of that body
of evidence supports the concept that deoxyribonucleotide biosynthesis for
bacteriophage T4 is carried out by an association of enzymes and other cellular
components in a complex called the dNTP synthetase complex. This dissertation
explores potential direct protein-protein interactions within this complex for the
preparation of pyrimidine deoxyribonucleotides.
Direct associations for enzymes involved in pyrimidine deoxyribonucleotide
biosynthesis were examined by affinity chromatography. It was determined that there
was a significant direct relationship between T4 thymidylate synthase and T4 dCMP
deaminase, between T4 dCTPase/dUTPase and T4 dCMP deaminase as well. The
interaction between thymidylate synthase and dCMP deaminase was significantly
influenced by the presence of dCTP, a positive effector of dCMP deaminase.
Furthermore, protein associations changed the kinetic character of pyrimidine
deoxyribonucleotide production. T4 dCTPase/dUTPase, a member of the dNTP
synthetase complex, significantly alters the kinetic nature of thymidylate synthase by
working with thymidylate synthase in a reciprocal relationship. T4 single-stranded
DNA binding protein, a member of the replication complex, alters the activity of
thymidylate synthase as well. Attempts to isolate a kinetically coupled complex from
two or more constituent proteins of the dNTP synthetase complex were frustrated by
protein degradation to fragments under 10 kDa in size.
Pyrimidine deoxyribonucleotide synthesis is located between the significant
energy investment of ribonucleotide reductase and phosphate attachments by kinases
to prepare the deoxyribonucleotide molecules for DNA replication. In bacteriophage
T4, intermediate reactions are driven by mass action but are modulated by subtleties
including direct protein associations and the presence of small molecules that
influence enzyme function. Through these and potentially similar controls, pools of
deoxyribonucleotides are prepared and delivered in a timely, balanced manner to the
DNA replication apparatus. / Graduation date: 2002
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Pyrimidine nucleotide biosynthesis in adult angiostrongylus Cantonensis (Nematoda : Metastrongyloidea) /So, Ngar-chung, Nellie. January 1993 (has links)
Thesis (Ph. D.)--University of Hong Kong, 1993. / Includes bibliographical references (leaves 164-199).
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Characterization of Pyrimidine Biosynthesis in Acinetobacter Calcoaceticus Using Wild Type and Mutant StrainsEntezampour, Mohammad 12 1900 (has links)
Pyrimidine nucleotide biosynthesis was studies in Acinetobacter calcoaceticus ADP-1. Pyrimidine auxotrophic mutants were isolated and characterized for this purpose. One such Pyr mutant, strain ADP-1-218 was chosen for further study.
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Isoorotate Decarboxylase Activity Present in Various Strains of NeurosporaHay, Kelly M. January 2000 (has links)
No description available.
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