RESUMEN
Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Proteínas Protozoarias , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citosol/enzimología , Cartilla de ADN/genética , Escherichia coli/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Protozoarios , Humanos , Cinética , Malato Deshidrogenasa/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
A cysteine proteinase, purified to homogeneity from epimastigotes of Trypanosoma cruzi, was strongly inhibited by L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64). The second-order rate constant was 20,800 M-1.s-1, and the reagent could be used for active site titration. The enzyme hydrolysed chromogenic peptides at the carboxyl Arg or Lys; it required at least one more amino acid, preferably Arg, Phe, Val or Leu, between the terminal Arg or Lys and the amino-blocking group. Enzyme activity on azocasein at pH 5.0 was increased by urea, maximal activity being attained at 2 M, and was still as active at 5 M urea as in its absence. Guanidine hydrochloride and KSCN also activated at low concentrations, but caused a strong inhibition above 2 M and 1 M, respectively. When azocasein was tested as a substrate at pH 7.0, there was no activation, and when synthetic substrates were used all chaotropic agents tested were inhibitory. The results suggest that the enzyme, for which we propose the trivial name 'cruzipain', differs in some aspects from all other cysteine proteinases described so far, although it shares several of the properties of mammalian cathepsin L.
Asunto(s)
Cisteína Endopeptidasas/aislamiento & purificación , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caseínas/metabolismo , Compuestos Cromogénicos/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática/efectos de los fármacos , Concentración de Iones de Hidrógeno , Cinética , Leucina/análogos & derivados , Leucina/farmacología , Datos de Secuencia Molecular , Proteínas Protozoarias , Especificidad por SustratoRESUMEN
The pyruvate kinase from Trypanosoma cruzi epimastigotes was activated by fructose 2,6-diphosphate ((A) 0.5 = 0.17 microM), through a decrease in (S) 0.5 and an increase in Vmax for both substrates. The enzyme was 50% inhibited by 0.9 mM ATP or 0.5 mM Pi in the presence of 30 mM MgCl2; these inhibitions were completely counteracted by 1.5 microM fructose 2,6-diphosphate. Both facts suggest that the effects are allosteric, and not due to chelation.