Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Thesis (Ph.D.)--McMaster University, 2003. / Advisor: Gerard Wright. Includes bibliographical references. Also available via World Wide Web.

Não Consta Resumo na Publicação / Abstracts Not Available

A importância na geração de oxaloacetato foi investigada através da determinação da atividade da piruvato carboxilase nos músculos estriados e da suplementação de seus precursores (aspartato e asparagina) na dieta de ratos. A atividade da piruvato carboxilase eleva-se durante o exercício físico e, portanto, deve fornecer mais oxaloacetato para a etapa inicial do ciclo de Krebs. A suplementação crônica (5 semanas) de aspartato e asparagina promove aumento da resistência ao esforço em ratos treinados em natação durante 1 hora diária por 5 semanas. Este efeito foi acompanhado de elevação no número e tamanho das mitocôndrias e alteração no metabolismo de glicose dos músculos esqueléticos (elevação do conteúdo de glicogênio e de sua síntese e diminuição da glicólise). Esses resultados sugerem que a geração de oxaloacetato desempenha papel fundamental na manutenção do esforço prolongado. A suplementação de aspartato e asparagina na dieta melhora a performance nessas condições, porém causa lesões na ultraestrutura muscular (mitocôndrias, linha \"Z\" e miofibrilas). / The importance of oxaloacetate formation was investigated by measuring pyruvate carboxylase activity in muscles and by given its precursors (aspartate, asparagine) in the diet of rats. The activity of pyruvate carboxylase markedly raised during physical effort and so might provide oxaloacetate for Krebs cycle functioning. The supplementation of aspartate and aspagine for a prolonged period of time (5 weeks) promotes increment in the resistance to exercise in rats trained to swimming during 1 hour daily for 5 weeks. This effect is accompanied by an increase in the size and number of mitochondria and also changes in glucose metabolism; elevation in glycogen synthesis and content and reduction in the rate of glycolysis. These results suggest that the production of oxaloacetate plays a role to maintain the moderate exercise during a prolonged period of time. Nevertheless, the aspartate and asparagine supplemented in the diet, despite improving the perfomance to moderate and prolonged exercise, provokes muscle ultraestructure lesions of mitochondria, \"Z\" line and miofibrils.

This study focused on the enzyme, aspartate transcarbamoylase (ATCase) from A. hydrophila, a Gram-negative bacterium found in fresh water. The molecular mass of the ATCase holoenzyme from A. hydrophila is 310 kDa. The enzyme is likely composed of 6 catalytic polypeptides of 34 kDa each and 6 regulatory polypeptides of 17 kDa each. The velocity-substrate curve for A. hydrophila ATCase is sigmoidal for both aspartate and carbamoylphosphate. The Km for aspartate was the highest to date for an enteric bacterium at 97.18 mM. The Km for carbamoylphosphate was 1.18 mM. When heated to 60 ºC, the specific activity of the enzyme dropped by more than 50 %. When heated to 100 ºC, the enzyme showed no activity. The enzyme's activity was inhibited by ATP, CTP or UTP.

Aspartate transcarbamoylase (ATCase) was purified from 16 selected bacterial species including existing Pseudomonas species and former species reassigned to new genera. An enormous diversity was seen among the 16 enzymes with each class of ATCase being represented. The smallest class, class C, with a catalytically active homotrimer, at 100 kDa, was found in Bacillus and other Gram positive bacteria. In this report, the ATCases from the Gram negatives, Shewanella putrefaciens and Stenotrophomonas maltophilia were added to class C membership. The enteric bacteria typify class B ATCases at 310 kDa, with a dodecameric structure composed of two catalytic trimers coupled to three regulatory dimers. A key feature of class B ATCases is the dissociability of the holoenzyme into regulatory and catalytic subunits which were enzymatically active. In this report, the ATCase from Pseudomonas indigofera was added to class B ATCases. The largest class, at 480 kDa, class A, contains the fluorescent Pseudomonas including most members of the 16S rRNA homology group I. Two polypeptides are produced from overlapping pyrBC' genes. The former, pyrB, encodes a 34 kDa catalytic polypeptide while pyrC' encodes a 45 kDa dihydroorotase-like polypeptide. Two non active trimers are made from six 34 kDa chains which are cemented by six 45 kDa chains to form the active dodecameric structure. Dissociation of the holoenyzme into its separate active subunits has not been possible. In this report, the ATCases from Comamonas acidovorans and C. testosteroni, were added to the class A enzymes. An even larger class of ATCase than class A at 600 kDa was discovered in Burkholderia cepacia. Stoichiometric measurements predict a dodecamer of six 39 kDa polypeptides and six 60 kDa polypeptides. Unlike other large pseudomonads ATCases, the enzyme from B. cepacia was dissociable into smaller active forms. Both the holoenzyme and its dissociated forms were regulated by nucleotide effectors. A new class of ATCase was proposed for B. cepacia type enzymes.

Bacterial aspartate transcarbamoylases (ATCase's) are divided into three classes that correspond to taxonomic relationships within the bacteria. The opportunistic pathogen Moraxeila catarrhalis has undergone several reclassifications based on traditional microbiological criteria. The previously uncharacterized ATCase from M. catarrhalis was purified to homogeneity and its chemical properties characterized. The ATCase from M. catarrhalis is a class C ATCase with an apparent molecular mass of 480-520 kDa. The M. catarrhalis ATCase is a dodecomer composed of six 35 kDa polypeptides and six 45 kDa polypeptides. The enzyme has an unusually high pH optimum of greater than pH 10. The enzyme exhibited hyperbolic kinetic with a Km for aspartate of 2 mM. A single, separate 78 kDa dihydroorotase from M. catarrhalis was identified and it was not associated with ATCase. These data support the reclassification of M. catarrhalis out of the Neisseriaceae family.

Aspartate transcarbamoylase (ATCase) of Pseudomonas putida consists of two different polypeptides, PyrB and PyrC' (Schurr et al, 1995). The role of the PyrC' and the assembly of PyrB and PyrC' have been studied. The ATCase made in vitro of P.putida PyrB with P.putida PyrC', and of E.coli PyrB with P.putida PyrC ' were generated under two different conditions, denaturation and renaturation, and untreated. It was found that PyrC' plays a role in the enzymatic regulation by ATP, CTP and UTP. In addition to playing a role in substrate binding, the PyrB polypeptide is also involved in effector binding (Kumar et al., manuscript in preparation). The most energetically preferred form of the P.putida WT is a dodecamer with a molecular mass of 480 kDa. The ratio between the PyrB and the PyrC' is 1:1. In studies of nucleotide binding, it was discovered that the P.putida PyrB was phosphorylated by a protein kinase in the cell extract. In the presence of 20 mM EDTA, this phosphorylation was inhibited and the inhibition could be overcome by the addition of divalent cations such as Zn2+ and Mg2+. This result suggested that the phosphorylation reaction required divalent cations. In the CAD complex of eukaryotes, phosphorylations of the CPSase and the linker region between ATCase and DHOase did not occur in the presence of UTP and it was hypothesized (Carrey, 1993) that UTP and phosphorylation(s) regulated the conformational change in the enzyme complex. Therefore, the same idea was approached with P.putida ATCase, where it was found that 1.0 mM UTP inhibited the phosphorylation of PyrB by more than 50%. These results suggested that the regulation of the conformational change of the P.putida ATCase might be similar to that of CAD. Furthermore, peptide mapping for phosphorylation sites was performed on P.putida ATCase WT, WT --11 amino acids and WT --34 amino acids from the N-terminus of the PyrB polypeptide. The results showed that the phosphorylation sites were located on the fragment that contained amino acid number-35 to amino acid number-112 from the N-terminus of the PyrB polypeptide.

Aspartate transcarbamoylase (ATCase) of Streptomyces was characterized and its interaction with other pyrimidine enzymes explored.

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.