N-Phosphorylated guanidino compounds, commonly referred to as phosphagens, play a critical role as an energy reserve because of the high energy phosphate that can be transferred when the renewal of ATP is needed. It has also been proposed that these compounds function in spatial buffering of cellular energy production sites. So, phosphagens act as reserves not only of ATP but also of inorganic phosphate, which is mostly returned to the medium by metabolic consumption of ATP. Phosphoarginine is the main reserve of high energy phosphate compounds in a wide variety of invertebrates. In addition phosphocreatine, phosphoglycocyamine, phosphotaurocyamine, phosphohypotaurocyamine, phosphoopheline, and phospholombricine are also found, whereas in vertebrates the only one present is phosphocreatine (1, 2).Arginine kinase (EC 2.7.3.3) is a member of a conserved family of phosphotransferases which also includes creatine kinase. These enzymes catalyze the reversible transfer of a phosphoryl group from ATP to a guanidino acceptor, which can be either an amino acid (e.g. lombricine or arginine) or a carboxylate (e.g. creatine or glycocyamine; Reaction 1).

Chagas' disease is a zoonosis caused by the parasite Trypanosoma cruzi, a haematic protozoan, transmitted by insects from the Reduviidae family. This constitutes a relevant health and socio-economic problem in the Americas, with 11 - 18 million people infected, and approximately 100 million people at risk. The therapeutic possibilities rely into two drugs, nifurtimox and benznidazole, that were discovered more than thirty years ago, and are mainly successful during the acute phase of the disease. In the majority of the cases the disease is diagnosed in the chronic phase, when the therapy is inefficient and the probability of cure is low. In addition, these drugs are highly toxic, with systemic side effects on patients. Trypanosoma cruzi has a metabolism largely based on the consumption of amino acids, mainly proline, aspartate and glutamate, which constitute the main carbon and energy sources in the insect stage of the parasite life cycle. These amino acids also participate in the differentiation process of the replicative non-infective form (epimastigote) to the non-replicative infective form (trypomastigote). In particular, the participation of proline in the intracellular differentiation cycle, which occurs in the mammalian host, was recently demonstrated. In addition, an arginine kinase has been described in T. cruzi and T. brucei, which converts free arginine to phosphoarginine, a phosphagen with a role as an energy reservoir. Arginine kinase seems to be an essential component of energy management during stress conditions. Taken together, these data indicate that amino acid metabolism may provide multiple as yet unexplored targets for therapeutic drugs.

A very specific L-arginine transporter showing high affinity has been characterized in Trypanosoma cruzi epimastigotes. Uptake was found to be dependent on L-arginine concentration and it was saturable. Values for maximum velocity and Km ranged between 48.1-57.5 pmol.min-1 per 3 x 10(7) cells and between 4.2-5.5 microM, respectively. The calculated activation energy and Q10 were 31.1 KJ.mol-1, and 1.7, respectively. Uptake velocity significantly increased when cells were preincubated in the absence of L-arginine. Cells retained the labeled amino acid independently of the presence or absence of exogenous L-arginine. The specificity of L-arginine uptake was demonstrated by competition assays in the presence of 80-fold molar excess of natural amino acids and several L-arginine derivatives. The highest levels of inhibition were caused by L-homoarginine, D-arginine, L-canavanine, L-ornithine, and L-citrulline. L-arginine uptake by T. cruzi epimastigotes was not affected by the presence of potassium or sodium ions in the incubation mixture or by pH changes in the range between 5.5-8.5. The major product of L-arginine uptake was characterized as phosphoarginine. Moreover, arginine kinase activity was detected in soluble extracts from T. cruzi epimastigotes.

Trypanosoma cruzi, the etiological agent of Chagas' disease, has a metabolism largely based on the consumption of glucose and proline. This amino acid is essential for host cells infection and intracellular differentiation. In this work we identified a proline transporter (TcAAAP069) by yeasts complementation assays and overexpression in Trypanosoma cruzi epimastigotes. TcAAAP069 is mono-specific for proline but presents an unusual feature; the lack of stereospecificity, because it is competitively inhibited by the D- enantiomer. Parasites overexpressing TcAAAP069 have an increased intracellular proline concentration, 2.6-fold higher than controls, as a consequence of a higher proline transport rate. Furthermore, augmented proline concentration correlates with an improved resistance to trypanocidal drugs and also to reactive oxygen species including hydrogen peroxide and nitric oxide, emulating natural physiological situations. The IC50s for nifurtimox, benznidazole, H2O2 and NO. were 125%, 68%, 44% and 112% higher than controls, respectively. Finally, proline metabolism generates a higher concentration (48%) of ATP in TcAAAP069 parasites. Since proline participates on essential energy pathways, stress and drug resistance responses, these results provide a novel target for the development of new drugs for the treatments for Chagas' disease.

A nitric oxide synthase was partially purified from soluble extracts of Trypanosoma cruzi epimastigote forms. The conversion of L-arginine to citrulline by this enzyme activity required NADPH and was blocked by EGTA. The reaction was activated by Ca2+, calmodulin, tetrahydrobiopterin, and FAD, and inhibited by N omega-methyl-L-arginine. L-Glutamate and N-methyl-D-aspartate stimulated in vivo conversion of L-arginine to citrulline by epimastigote cells. These stimulations could be blocked by EGTA, MK-801, and ketamine and enhanced by glycine. A sodium nitroprusside-activated guanylyl cyclase activity was detected in cell-free, soluble preparations of T. cruzi epimastigotes. L-Glutamate, N-methyl-D-aspartate, and sodium nitroprusside increased epimastigote cyclic GMP levels. MK-801 bound specifically to T. cruzi epimastigote cells. This binding was competed by ketamine and enhanced by glycine or L-serine. Evidence thus indicates that in T. cruzi epimastigotes, L-glutamate controls cyclic GMP levels through a pathway mediated by nitric oxide.

Arginine kinase catalyzes the reversible transphosphorylation between adenosine diphosphate (ADP) and phosphoarginine, which is involved in temporal and spatial adenosine triphosphate (ATP) bu¡ering. Here we demonstrate that the homologous overexpression of the Trypanosoma cruzi arginine kinase improves the ability of the transfectant cells to grow and resist nutritional and pH stress conditions. The stable transfected parasites showed an increased cell density since day 10 of culture, when the carbon sources became scarce, which resulted 2.5-fold higher than the control group on day 28. Additional stress conditions were also tested. We propose that arginine kinase is involved in the adaptation of the parasite to environmental changes. ß

Adenylate kinases occur classically as cytoplasmic and mitochondrial enzymes, but the expression of seven adenylate kinases in the flagellated protozoan parasite Trypanosoma brucei (order, Kinetoplastida; family, Trypanosomatidae) easily exceeds the number of isoforms previously observed within a single cell and raises questions as to their location and function. We show that a requirement to target adenylate kinase into glycosomes, which are unique kinetoplastid-specific microbodies of the peroxisome class in which many reactions of carbohydrate metabolism are compartmentalized, and two different flagellar structures as well as cytoplasm and mitochondrion explains the expansion of this gene family in trypanosomes. The three isoforms that are selectively built into either the flagellar axoneme or the extra-axonemal paraflagellar rod, which is essential for motility, all contain long N-terminal extensions. Biochemical analysis of the only short form trypanosome adenylate kinase revealed that this enzyme catalyzes phosphotransfer of ␥-phosphate from ATP to AMP, CMP, and UMP acceptors; its high activity and specificity toward CMP is likely to reflect an adaptation to very low intracellular cytidine nucleotide pools. Analysis of some of the phosphotransfer network using RNA interference suggests considerable complexity within the homeostasis of cellular energetics. The anchoring of specific adenylate kinases within two distinct flagellar structures provides a paradigm for metabolic organization and efficiency in other flagellates.

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