The present study has resulted in the development of a procedure for the specific chemical fragmentation of human phosphoglucose isomerase into a minimal number of peptides. A two-cycle procedure for cleaving the protein with 2-nitro-5- thiocyanobenzoic acid results in four primary peptides and three overlap peptides. The peptides can be readily separated on the basis of their size by using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary peptide alignments have been considered, and amino acid analyses have been performed. End-terminal analyses of the enzyme revealed a carboxyl terminal sequence of Asp-Val-Gln and a blocked amino terminus. The cysteine cleavage procedure provides an excellent method for the identification and location of specific genetic mutations of human phosphoglucose isomerase.

Structural properties of phosphoglucose isomerases isolated from a variety of species have been compared by peptide fingerprinting, predicted amino acid sequence homologies and by denaturation and renaturation studies. The enzymes are more readily denatured in guanidinium chloride than in urea, and the isomerase isolated from yeast is more stable toward acid pH than the rabbit muscle enzyme. The rates of guanidinium chloride-induced denaturation are markedly increased by ionic strength and decreased by substrates, competitive inhibitors or glycerol. The enzyme can be renatured, but only in the presence of glycerol. The renaturation process is dependent on protein concentration and temperature and provides a method for the formation of mixed species heterodimers.

A procedure was developed for the rapid isolation of human phosphoglucose isomerase by substrate-induced elution from cellulose phosphate. The high degree of selectivity of the elution provided homogenous enzyme from erythrocytes after a purification of approximately 30,000-fold with a recovery of approximately 70%. The enzyme was also isolated from other human tissues by a similar procedure.

A method for purification and radiolabelling phosphoglucose isomerase was devised in order to develop a sensitive quantitative radioimmunoassay for the detection of the enzyme irrespective of its catalytic activity. For four genetic variants of PGI no difference in the molecular specific activity was observed. In one variant (PGI-Denton), liver and heart tissue extracts, and in mature erythrocytes (as compared to normal erythrocytes), a decreased molecular specific activity was observed which initially may imply that these samples contain cross-reactive material which is not catalytically active.

A tripanossomose africana, ou doença do sono, como é popularmente conhecida, atingiu 25 mil pessoas só em 1995. Estima-se que a doença seja responsável pela morte de 10.000 pessoas a cada ano, segundo os dados da Organização Mundial da Saúde. Sua distribuição é exclusivamente africana, devido ao fato de sua transmissão depender da Tsé-Tsé, mosca do gênero Glossina que encontra lá condições favoráveis para sua proliferação. Atualmente os medicamentos existentes não são eficientes e devem ser ministrados em doses altas, provocando graves efeitos colaterais. O parasita Tripanosoma brucei, na forma encontrada no sangue dos mamíferos (tripomastigota), é o responsável pela doença e, assim como toda a família Trypanosomatidea, é altamente dependente de glicose. Esses parasitas consomem a quantidade de glicose equivalente à sua massa em aproximadamente sete horas. A via glicolítica torna-se, portanto, chave para o desenvolvimento de inibidores que possam ser utilizados no combate a estes parasitas. Nesse sentido, temos realizado estudos com a enzima glicose-6-fosfato isomerase (glucose-6-fosfato isomerase; Phosphoglucose Isomerase PGI; EC 5.3.1.9), responsável pela isomerização reversível da D-glicose-6-fosfato e D-frutose-6-fosfato e participa na glicólise como a segunda enzima da via. Os trabalhos realizados até aqui culminaram na sua expressão em forma recombinante, purificação através de coluna de afinidade, e caracterizações enzimáticas. Sua atividade específica foi determinada através de métodos já estabelecidos encontrados na literatura. O IC50 da enzima frente a quatro inibidores da reação foi determinado. O trabalho finaliza com a construção de um modelo estrutural da enzima determinado através de métodos de modelagem molecular por homologia. / The African trypanosomiasis, or sleeping sickness as is popularly known, affected 25 thousand people only in 1995. It is estimated that the disease is responsible for 10 thousand deaths per year, according to data provided from the World Health Organization (WHO). The distribution of the disease is exclusively African due to the transmission being dependent on the tsé-tsé vector. A fly, belonging to the Glossinia genus, finds in the African continent favorable conditions for its proliferation. Presently the existing drugs are not efficient and have to be applied in high dosage, resulting in severe side effects. The bloodstream form (tripomastigote) of the parasite Trypanosoma brucei is responsible for the disease and such as the whole Trypanosomatidae family is dependent on glucose. Those parasites consume a quantity of glucose equivalent to its mass in approximately seven hours. This characteristic results in the glycolitic pathway been a key target for drug development against those parasites. In this direction we are developing research with the enzyme glucose-6-phosphate isomerase (Phosphoglucose lsomerase PGI; EC 5.3.1.9) responsible for the reversible isomnerisation of D-glucose6-phosphate and D-fuctcose-6-phosphate. PGI participates as the second enzyme in the glycolytic pathway. The work developed so far resulted in the expression of the recombinant form of the parasite PGI, its affinity purification and enzimatic characterization. The specific activity was determined with established methods. The IC50 of four inhibitors was determined and a structural model of T brucei PGI was built by molecular modeling techniques.

A tripanossomose africana, ou doença do sono, como é popularmente conhecida, atingiu 25 mil pessoas só em 1995. Estima-se que a doença seja responsável pela morte de 10.000 pessoas a cada ano, segundo os dados da Organização Mundial da Saúde. Sua distribuição é exclusivamente africana, devido ao fato de sua transmissão depender da Tsé-Tsé, mosca do gênero Glossina que encontra lá condições favoráveis para sua proliferação. Atualmente os medicamentos existentes não são eficientes e devem ser ministrados em doses altas, provocando graves efeitos colaterais. O parasita Tripanosoma brucei, na forma encontrada no sangue dos mamíferos (tripomastigota), é o responsável pela doença e, assim como toda a família Trypanosomatidea, é altamente dependente de glicose. Esses parasitas consomem a quantidade de glicose equivalente à sua massa em aproximadamente sete horas. A via glicolítica torna-se, portanto, chave para o desenvolvimento de inibidores que possam ser utilizados no combate a estes parasitas. Nesse sentido, temos realizado estudos com a enzima glicose-6-fosfato isomerase (glucose-6-fosfato isomerase; Phosphoglucose Isomerase PGI; EC 5.3.1.9), responsável pela isomerização reversível da D-glicose-6-fosfato e D-frutose-6-fosfato e participa na glicólise como a segunda enzima da via. Os trabalhos realizados até aqui culminaram na sua expressão em forma recombinante, purificação através de coluna de afinidade, e caracterizações enzimáticas. Sua atividade específica foi determinada através de métodos já estabelecidos encontrados na literatura. O IC50 da enzima frente a quatro inibidores da reação foi determinado. O trabalho finaliza com a construção de um modelo estrutural da enzima determinado através de métodos de modelagem molecular por homologia. / The African trypanosomiasis, or sleeping sickness as is popularly known, affected 25 thousand people only in 1995. It is estimated that the disease is responsible for 10 thousand deaths per year, according to data provided from the World Health Organization (WHO). The distribution of the disease is exclusively African due to the transmission being dependent on the tsé-tsé vector. A fly, belonging to the Glossinia genus, finds in the African continent favorable conditions for its proliferation. Presently the existing drugs are not efficient and have to be applied in high dosage, resulting in severe side effects. The bloodstream form (tripomastigote) of the parasite Trypanosoma brucei is responsible for the disease and such as the whole Trypanosomatidae family is dependent on glucose. Those parasites consume a quantity of glucose equivalent to its mass in approximately seven hours. This characteristic results in the glycolitic pathway been a key target for drug development against those parasites. In this direction we are developing research with the enzyme glucose-6-phosphate isomerase (Phosphoglucose lsomerase PGI; EC 5.3.1.9) responsible for the reversible isomnerisation of D-glucose6-phosphate and D-fuctcose-6-phosphate. PGI participates as the second enzyme in the glycolytic pathway. The work developed so far resulted in the expression of the recombinant form of the parasite PGI, its affinity purification and enzimatic characterization. The specific activity was determined with established methods. The IC50 of four inhibitors was determined and a structural model of T brucei PGI was built by molecular modeling techniques.

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.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Glycolysis is the main metabolic route in Saccharomyces cerevisiae and it is the sequence of enzyme catalyzed reactions that oxidatively convert glucose to pyruvic acid in the yeast cytoplasm. In addition to the basic steps, glycolysis involves branch points providing the intermediary building blocks of the cell (i.e amino acids and nucleotides). One of these pathways is glucose-6-phosphate branch point which is a junction of glycolytic pathway and pentose phosphate pathway. At this point glucose-6-phosphate can be converted to fructose-6-phosphate a metabolite of glycolytic pathway by phosphoglucoisomerase or it can be dehydrogenated to 6-phosphogluconolactone by glucose-6-phosphate dehydrogenase which is the first enzyme of the pentose phosphate pathway. In this study, the influence of different nitrogen sources on the flux distribution through the pentose phosphate pathway and glycolysis in Saccharomyces cerevisiae was examined. For this purpose, four different compositions of nitrogen sources were used in growth media. The growth medium contained one of the following composition of nitrogen sources / only ammonium sulfate, only yeast nitrogen base, ammonium sulfate and histidine, yeast nitrogen base and histidine. Histidine was added because its synthesis branches from pentose phosphate pathway. In order to analyse the effect of the different compositions of nitrogen sources on the physiology of the yeast, specific activities of hexokinase, phosphoglucose isomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase enzymes were measured in the crude extracts of the biomass samples taken in the late exponential phase of the cultures. Addition of histidine caused an increase in the specific activities of all the enzymes analysed in medium containing ammonium sulfate. The specific activity of hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase in medium containing yeast nitrogen base and histidine were higher than medium containing yeast nitrogen base. However, the specific activity of 6-phosphogluconate dehydrogenase decreased 3.1% in medium containing yeast nitrogen base and histidine medium with respect to medium with only yeast nitrogen base. The OD value and dry weight in the culture containing histidine aminoacid was higher than the cultures contaning only ammonium sulfate and only yeast nitrogen base. Also the period of the exponential phase was shorter in medium containing ammonium sulfate and histidine and yeast nitrogen base and histidine than medium only ammonium sulfate and only yeast nitrogen base.

Les aldose-cétose isomérases sont des enzymes catalysant l’isomérisation réversible entre un aldose et un cétose. Nous avons étudiés trois d’entre-elles : les phosphoribose isomérases (RPI), les phosphomannose isomérases (PMI) et les phosphoglucose isomérases (PGI). Ces enzymes interviennent dans différentes voies métaboliques comme la glycolyse, la néoglucogenèse, la voie des pentoses phosphates ou le métabolisme du mannose. Il a été montré qu’elles jouent un rôle important pour assurer la survie et le développement de plusieurs parasites responsables de maladies comme la leishmaniose, la mucoviscidose, la tuberculose, le paludisme ou la maladie du sommeil. Ces enzymes sont donc des cibles thérapeutiques potentielles. Ainsi, les puissants inhibiteurs de ces enzymes peuvent donc être des agents thérapeutiques efficaces pour combattre ces maladies. Les réactions catalysées par ces enzymes impliquent des intermédiaires de haute énergie (IHE) de type 1,2-cis-ènediol(ate). La synthèse d’analogues de ces intermédiaires a permis d’obtenir au laboratoire, les meilleurs inhibiteurs connus de ces enzymes, l’acide 5-phospho-D-arabononohydroxamique (5PAH, meilleur inhibiteur des PMI et PGI) et le 5-phospho-D-ribonate (5PRA, meilleur inhibiteur des RPI). Cependant, ces inhibiteurs possèdent une fonction phosphate facilement hydrolysable en milieu physiologique. Ce qui les rend inactifs in vivo. Au cours de ce travail de thèse, des phosphoanalogues du 5PAH, du 5-phospho-D-ribose (R5P, le substrat des RPI) et du 5PRA possédant une fonction malonate, phosphonate, phosphorothiate, sulfate et sulfonate à la place de la fonction phosphate ont été obtenus par des voies de synthèse multi-étapes faisant intervenir le D-arabinose ou le D-ribose comme produit de départ. Les propriétés inhibitrices de ces composés ont ensuite été déterminées et leur stabilité en milieu physiologique évaluée. Le phosphoanalogue du 5PAH de type malonate, l’acide 5-désoxy-5-dicarboxyméthyl-D-arabinonohydroxamique (5DCAH) est un inhibiteur moyen et stable de la PMI d’Escherichia Coli. Parmi les phosphoanalogues du R5P, les composés de type sulfate et sulfonate, respectivement, le 5-sulfate-D-ribose (5SR) et 5-désoxy-5-sulfonométhyl-D-ribose (5SMR) sont de bons inhibiteurs de trois RPI (la RPI d’épinard, la RPI d’Escherichia Coli et la RPI de Micobacterium tuberculosis). Seul le composé de type sulfonate est stable en milieu physiologique. Le phosphoanalogue de type malonate, le 5-désoxy-5-dicarboxyméthyl-D-ribose (5DCR) est un inhibiteur moyen de ces trois RPI. En revanche, les phosphoanalogues de type phosphorothioate et phosphonate, respectivement, le 5-désoxy-5-phosphorothioate-D-ribose (5PTR) et 5-désoxy-5-phosphonométhyl-D-ribose (5PMR) sont de mauvais inhibiteurs. Le phosphoanalogue de type phosphonate du 5PRA, le 5-désoxy-5-phosphonométhyl-D-ribonate (5PMRA) est un bon inhibiteur de la RPI de Micobacterium tuberculosis. De plus, ce composé est stable en milieu physiologique. Il est en revanche un mauvais inhibiteur de la RPI d’épinard et d’Escherichia Coli. Ces résultats sont particulièrement prometteurs puisque le 5PMRA est à ce jour le meilleur inhibiteur stable et spécifique de la RPI de Micobacterium tuberculosis. / Aldose-ketose isomerases are enzymes which catalyze the interconversion of an aldose and a ketose. We have studied three of them: phosphoribose isomerase (RPI), phosphomannose isomerase (PMI) and phosphoglucose isomerase (PGI). These enzymes play a major role in various metabolic pathways as glycolysis, neoglucogenesis, the pentoses phosphates pathways or the mannose metabolism. It has been shown to have a crucial role for the survival and development of several microorganisms responsible for diseases as the leishmaniose, the cystic fibrosis, the tuberculosis, the malaria or the insomnia. These enzymes are thus potential therapeutic targets. Consequently, strong inhibitors of these enzymes could provide efficient therapeutic tools against these deseases. The reactions catalyzed by these enzymes involve intermediaries of high energy (IHE) of 1,2-cis-enediol(ate) type. The synthesis of analogues of these intermediaries allowed to obtain in the laboratory, the best inhibitors known for these enzymes, the acid 5-phospho-D-arabononohydroxamique (5PAH, the best inhibitor of the PMI and PGI) and the 5-phospho-D-ribonate (5PRA, the best inhibitor of the RPI). However, these inhibitors possess a phosphate group which is easily hydrolysable in physiological environment, what makes them inactive in vivo. During this work of thesis, phosphoanalogues of the 5PAH, the 5-phospho-D-ribose (R5P, the substrate of the RPI) and of the 5PRA possessing a malonate, phosphonate, phosphorothiate, sulphate and sulfonate were obtained by multi-steps synthesis bringing in D-arabinose or D-ribose as starting product. The inhibitive properties of these compounds were then determined and their stability in physiological environment evaluated. The phosphoanalogue of the 5PAH of malonate type, the acid 5-desoxy-5-dicarboxyméthyl-D-arabinonohydroxamique (5DCAH) is a modest and stable inhibitor of the PMI of Escherichia Coli. Among the phosphoanalogues of the R5P, the compounds of sulphate and sulfonate types, respectively, the 5-sulfate-D-ribose (5SR) and 5-desoxy-sulfonomethyl-D-ribose (5SMR), are good inhibitors of three RPI (the RPI of spinach, the RPI of Escherichia Coli and the RPI of Micobacterium tuberculosis). Only the compound of sulfonate type is stable in physiological environment. The phosphoanalogue of malonate type, the 5-desoxy-5-dicarboxymethyl-D-ribose (5DCR) is a modest inhibitor of this three RPI. On the other hand, the phosphoanalogues of phosphorothioate and phosphonate types, respectively, the 5-desoxy-5-phosphorothioate-D-ribose (5PTR) and the 5-desoxy-5-phosphonomethyl-D-ribose (5PMR), are bad inhibitors. The phosphoanalogue of phosphonate type of the 5PRA, the 5-desoxy-5-phosphonomethyl-D-ribonate (5PMRA), is a good inhibitor of the RPI of Micobacterium tuberculosis. Furthermore, this compound is stable in physiological environment. It is on the other hand a bad inhibitor of the RPI of spinach and Escherichia Coli. These results are particularly promising because the 5PMRA is this day the best stable and specific inhibitor of the RPI of Micobacterium tuberculosis.