RESUMEN
Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Lisosomas/enzimología , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Trypanosoma cruzi/genéticaAsunto(s)
Antígenos de Protozoos/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Glicoproteínas/metabolismo , Trypanosoma cruzi/enzimología , Animales , Cumarinas/farmacología , Cisteína Endopeptidasas/efectos de los fármacos , Dipéptidos/farmacología , Relación Dosis-Respuesta a Droga , Glicoproteínas/antagonistas & inhibidores , Calor , Concentración de Iones de Hidrógeno , HidrólisisRESUMEN
Kinetic and structural studies have been carried out of two isoenzymes of hexokinase from the rat, hexokinase II and glucokinase. Although both enzymes are monomeric, hexokinase II has a molecular weight double that of glucokinase and resembles a dimer of glucokinase. The co-operativity of glucokinase, which is not observed for hexokinase II, appears to be kinetic in origin rather than the consequence of ineractions between distinct glucose-binding sites.