RESUMO
Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite.
Assuntos
Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Trypanosoma/enzimologia , Animais , Digitonina/farmacologia , Glucosefosfato Desidrogenase/isolamento & purificação , Glucosefosfato Desidrogenase/metabolismo , Glicólise , Hexoquinase/isolamento & purificação , Hexoquinase/metabolismo , Cavalos , Indicadores e Reagentes/farmacologia , Malato Desidrogenase/isolamento & purificação , Malato Desidrogenase/metabolismo , Camundongos , Microcorpos/enzimologia , Microscopia de Fluorescência , Permeabilidade/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/isolamento & purificação , Fosfoglicerato Quinase/isolamento & purificação , Fosfoglicerato Quinase/metabolismo , Fosfopiruvato Hidratase/isolamento & purificação , Fosfopiruvato Hidratase/metabolismo , Piruvato Ortofosfato Diquinase/isolamento & purificação , Coelhos , Ratos , Ratos Wistar , Trypanosoma/efeitos dos fármacosRESUMO
The parasite Entamoeba histolytica is an organism whose main energetic source comes from glycolysis. It has the singularity that several of its glycolytic enzymes use pyrophosphate as an alternative phosphate donor. Thus, pyruvate phosphate dikinase (PPDK), an inorganic pyrophosphate (PPi)-dependent enzyme, substitutes pyruvate kinase present in humans. We previously cloned and sequenced the gene that codifies for PPDK in E. histolytica. We now report its expression in a bacterial system and its purification to 98% homogeneity. We determined its K(m) for phosphoenolpyruvate, AMP and PPi (21, < 5 and 100 microM, respectively). Unlike PPDK from maize and bacteria and pyruvate kinase from other cells, EhPPDk is dependent on divalent cations but does not require monovalent cations for activity. The enzyme has an optimum pH of 6.0, it is labile to low temperatures and has a tetrameric structure. Since EhPPDK is a PPi-dependent enzyme, we also tested the effect of some pyrophosphate analogs as inhibitors of activity. Studies on the function and structure of this enzyme may be important for therapeutic research in several parasitic diseases, since it has no counterpart in humans.