Carbamoyl phosphate synthetase (CPS) catalyzes the formation of carbamoyl phosphate (CP) from MgATP, bicarbonate, and glutamine. It has three active sites, one present on the small subunit and the two phosphorylation sites present on the large subunit. These two nucleotide binding sites are homologous. Six compounds were designed to mimic the reactive intermediate species carboxy phosphate, and product cabamoyl phosphate. The apparent Ki values calculated estimated the inhibitory strengths of these compounds. These plots were also utilized in identifying the linear inhibitors, nonlinear inhibitors and partial inhibitors. Inhibition patterns were obtained with these compounds using various assay formats. Partial inhibition displayed by phosphono formate for the full biosynthetic reaction can be utilized in support of the sequential mechanism for CPS.

Carbamoyl phosphate synthetase (CPS) from E. coli catalyzes the formation of carbamoyl phosphate, an intermediate in the biosynthesis of pyrimidine nucleotides and arginine, from glutamine, bicarbonate and two molecules of MgATP. This reaction is catalyzed by three separate active sites that are separated in space by ~100 Å. The transfer of ammonia and carbamate through the two intramolecular tunnels was investigated by molecular dynamics simulations and experimental characterization of mutations within. The presence of an unstable reaction intermediate, carboxyphosphate, was established. A method for studying the synchronization of the two active sites on the large subunit of CPS was developed. The potential of mean force (PMF) calculations along the ammonia and carbamate transfer pathways indicate a low free-energy path for the translocation of ammonia. The highest barrier for ammonia is 7.2 kcal/mol which corresponds to a narrow turning gate surrounded by the side chains of Cys-232, Ala-251, and Ala-314 in the large subunit. A blockage in the passageway was introduced by the triple mutant C232V/A251V/A314V, which was unable to synthesize carbamoyl phosphate. The release of phosphate is necessary for the injection of carbamate into the carbamate tunnel. Two mutants, A23F and G575F, were designed to block the migration of carbamate through carbamate tunnel. The mutants retained only 1.7 percent and 3.8 percent of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild-type CPS, respectively. Formate can be utilized by CPS in the absence of bicarbonate to form formyl phosphate. This intermediate was observed by 31P, 13C, and 1H NMR. For the three NMR methods a peak corresponding to formyl phosphate was observed at 2.15 ppm (31P) , 162.4 ppm (13C), and 8.39 and 7.94 ppm (1H). The rate of formation of formyl phosphate is 0.025 ± 0.005 s-1. Formamide was not detected in the presence of an ammonia source. Fluorescence anisotropy measurements on the C551A/S171C and C551A/S717C mutants provided insight into a possible mechanism of synchronization between the two active sites on the large subunit. The biggest fluorescence anisotropy change was observed at the N-terminal domain in the presence of AMPPNP and ATP.

This thesis describes the use of functional genomics screens in yeast to study anticancer drug action and intracellular transport. The yeast Saccharomyces cerevisiae provides a particularly useful model system for global drug screens, due to the availability of knockout mutants for all yeast genes. A complete collection of yeast deletion mutants was screened for sensitivity to monensin, a drug that affects intracellular transport. A total of 63 deletion mutants were recovered, and most of them were in genes involved in transport beyond the Golgi. Surprisingly, none of the V-ATPase subunits were identified. Further analysis showed that a V-ATPase mutant interacts synthetically with many of the monensin-sensitive mutants. This suggests that monensin may act by interfering with the maintenance of an acidic pH in the late secretory pathway. The second part of the thesis concerns identification of the underlying causes for susceptibility and resistance to the anticancer drug 5-fluorouracil (5-FU). In a functional genomics screen for 5-FU sensitivity, 138 mutants were identified. Mutants affecting tRNA modifications were particularly sensitive to 5-FU. The cytotoxic effect of 5-FU is strongly enhanced in these mutants at higher temperature, which suggests that tRNAs are destabilized in the presence of 5-FU. Consistent with this, higher temperatures also potentiate the effect of 5-FU on wild type yeast cells. In a plasmid screen, five genes were found to confer resistance to 5-FU when overexpressed. Two of these genes, CPA1 and CPA2 encode the two subunits of the arginine-specific carbamoyl-phosphate synthase. The three other genes, HMS1, YAE1 and YJL055W are partially dependent on CPA1 and CPA2 for their effects on 5-FU resistance. The specific incorporation of [14C]5-FU into tRNA is diminished in all overexpressor strains, which suggest that they may affect the pyrimidine biosynthetic pathway.

Thesis (M.S.)--Miami University, Dept. of Zoology, 2007. / Title from first page of PDF document. Includes bibliographical references (p. 17-19).

Stuttgart, Univ., Diss., 2001.

Universiẗat, Diss., 2001--Stuttgart.

BACKGROUND: After proteolysis, the majority of released amino acids from dietary protein are transported to the liver for gluconeogenesis or to peripheral tissues where they are used for protein synthesis and eventually catabolized, producing ammonia as a byproduct. High ammonia levels in the brain are a major contributor to the decreased neural function that occurs in several pathological conditions such as hepatic encephalopathy when liver urea cycle function is compromised. Therefore, it is important to gain a deeper understanding of human ammonia metabolism. The objective of this study was to predict changes in blood ammonia levels resulting from alterations in dietary protein intake, from liver disease, or from partial loss of urea cycle function. METHODS: A simple mathematical model was created using MATLAB SimBiology and data from published studies. Simulations were performed and results analyzed to determine steady state changes in ammonia levels resulting from varying dietary protein intake and varying liver enzyme activity levels to simulate liver disease. As a toxicity reference, viability was measured in SH-SY5Y neuroblastoma cells following differentiation and ammonium chloride treatment. RESULTS: Results from control simulations yielded steady state blood ammonia levels within normal physiological limits. Increasing dietary protein intake by 72% resulted in a 59% increase in blood ammonia levels. Simulations of liver cirrhosis increased blood ammonia levels by 41 to 130% depending upon the level of dietary protein intake. Simulations of heterozygous individuals carrying a loss of function allele of the urea cycle carbamoyl phosphate synthetase I (CPS1) gene resulted in more than a tripling of blood ammonia levels (from roughly 18 to 60 μM depending on dietary protein intake). The viability of differentiated SH-SY5Y cells was decreased by 14% by the addition of a slightly higher amount of ammonium chloride (90 μM). CONCLUSIONS: Data from the model suggest decreasing protein consumption may be one simple strategy to decrease blood ammonia levels and minimize the risk of developing hepatic encephalopathy for many liver disease patients. In addition, the model suggests subjects who are known carriers of disease-causing CPS1 alleles may benefit from monitoring blood ammonia levels and limiting the level of protein intake if ammonia levels are high.

No description available.

Molecular aspects of nitrogen metabolism in vertebrates is an interesting area of physiology and evolution to explore due to the different ways in which animals excrete nitrogenous waste as they transition from an aquatic to a terrestrial lifestyle. Two main products of nitrogen metabolism in fishes are ammonia and urea. Ammonia is produced during protein catabolism and build up of ammonia is toxic. Some aquatic vertebrates convert ammonia into a less toxic compound urea via de novo synthesis through the ornithine-urea cycle (O-UC). Five enzymes are involved in the O-UC: carbamoyl phosphate synthetase (CPS), ornithine carbamoyl transferase (OCT), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (ARG). An accessory enzyme, glutamine synthetase (GS) also participates in the "fish-type" O-UC. Teleosts excrete ammonia passively over their gills into the aquatic environment. The teleost, Opsanus beta, has been shown to increase urea production after 48 hours of crowding. This thesis explored how crowding stress affected nitrogen metabolite levels of ammonia and urea and O-UC gene expression and enzyme activity in O. beta. Lungfishes while in an aquatic environment avoid ammonia toxicity by releasing excess ammonia across their gills, but when stranded on land they produce urea through the O-UC. Urea production via the O-UC has a metabolic cost of at least four ATP molecules. This thesis explored the response of a lungfish, Protopterus annectens, to six days of aerial exposure and re-immersion conditions by measuring concentrations of O-UC mRNA expression and enzyme activity and nitrogen metabolites ammonia and urea. CPS acts as the entry point to the O-UC and based on enzymatic studies, most aquatic vertebrates utilize one isoform of this enzyme (CPSIII) while terrestrial vertebrates utilize a different isoform of this enzyme (CPSI). Lungfishes are a particularly interesting group of air-breathing fishes, not only because of their link to the origins of tetrapods, but also because CPS I may have originated within this group. Both CPS III and CPS I have been enzymatically described within this group. This thesis uses phylogenetics to investigate how CPS nucleotide sequences in lungfishes evolved compared to other vertebrates.

Made available in DSpace on 2016-08-17T18:39:31Z (GMT). No. of bitstreams: 1 2631.pdf: 1610800 bytes, checksum: 9f443d37247fb5fee135a70ec93a8142 (MD5) Previous issue date: 2008-09-29 / Financiadora de Estudos e Projetos / Immobilization and stabilization of enzymes increases their potential for use in industrial scale. D-hydantoinases (dihidropirimidina amidrohidrolase EC 3.5.2.2) catalyze the hydrolysis of D-hydantoins, generating the corresponding Ncarbamoil- D-amino acid and are used in the production of D-amino acids, including Dphenylglycine and D-p-hydroxyphenylglycine.This work reports studies for immobilization and stabilization of D-hydantoinase from Vigna angularis (E.C. 3.5.2.2.). Different strategies of multipoint covalent attachment in organic supports as chitosan and agarose were used. Different protocols of immobilization were employed, being the adittion of ions during the reduction step with the NaBH4 important to protect enzyme catalytic site. The active and stabilized derivatives were used to catalyze the hydrolysis of D-phenylhydantoin. The temperature and pH enzyme profiles showed maximum enzyme activity at 60ºC and pH 10,0. The subunits of the enzyme present molecular mass aroundt 50kDa. The enzyme immobilized in glyoxyl-agarose in the presence of Zn2+ ions during the reduction step, with immobilization time of 24h, was the best derivative, being 89-fold more stable than the soluble enzyme. The analysis of amino acids showed that a 50% of lysines residue present in the enzymes was covalently linked in glyoxyl-agarose. The enzyme immobilized in epoxy-chitosan-alginate was 20-fold more stable than the soluble enzyme. All the tested immobilization protocols led to 100% of immobilization yield. Soluble enzyme and the best glyoxyl and chitosan enzyme derivatives were used to catalyze the hydrolysis of D- phenylhydantoin , and led to the production of 99% of NCarbamoil- D-Phenylglycine after 3, 9 and 15h of reaction respectively. / A imobilização e estabilização de enzimas aumentam muito o potencial de uso industrial desses catalisadores. D-hidantoinases (dihidropirimidina amidrohidrolase EC 3.5.2.2) são enzimas que catalisam a hidrólise de hidantoínas, com abertura do anel, para o correspondente N-carbamoil-D-aminoácido e são usadas na produção de Daminoácidos, incluindo D-fenilglicina e D-p-hidroxifenilglicina. Este trabalho relata os estudos desenvolvidos para a imobilização e estabilização de D-hidantoinase de Vigna angularis (3.5.2.2.). Foram abordadas diferentes estratégias de imobilização multipontual em suportes orgânicos como quitosana e agarose. Diferentes protocolos de imobilização foram empregados, sendo adição de íons durante a redução com NaBH4 importante para proteção do centro catalítico da enzima. Os derivados ativos e estabilizados foram empregados na reação de hidrólise da fenilhidantoína. O estudo de temperatura e pH de máxima atividade da enzima foi 60°C e pH 10,0. As subunidades da enzima apresentam peso molecular, com valor próximo a 50kDa. A enzima imobilizada em glioxil-agarose na presença dos íons Zn2+ durante a etapa de redução, com tempo de imobilização de 24 h foi o derivado mais estável sendo 89 vezes mais estável que a enzima solúvel. A análise de aminoácidos mostrou que aproximadamente 50% dos resíduos de lisina presentes na enzima foram covalentemente ligados no derivado de glioxil-agarose. A enzima imobilizada em quitosana-alginato-epoxilado foi 20 vezes mais estável que a enzima solúvel. Todos os procedimentos de imobilização testados levaram a 100% de rendimento de imobilização. Enzima solúvel e os melhores derivados obtidos por imobilização em glioxil e quitosana foram usados na catálise da hidrólise de fenilhidantoína, produzindo 99% de N-Carbamoil-D-fenilglicina nos tempos de 3, 9 e 15 h, respectivamente.