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|Title:||Propriedades hidrolíticas de cisteíno-peptidases recombinantes de Tripanossomatídeos|
|Other Titles:||Hidrolytic properties of recombinant cysteine peptidases from Trypanosomatides|
|Authors:||Chagas, Jair Ribeiro [UNIFESP]|
Judice, Wagner Alves de Souza [UNIFESP]
|Publisher:||Universidade Federal de São Paulo (UNIFESP)|
|Citation:||São Paulo: [s.n.], 2004. 140 p.|
|Abstract:||Trypanosoma cruzi cysteine proteinase cruzipain contains a 130-aminoacid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannone-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2' subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp. Large differences in the kinetic parameters were not observed between the enzymes; however, Km, values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH-activity profile between the two enzymes was found, but in 1 M NaCl cruzipain presented a kcat values significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k-1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis-Menten parameters kcat /Km, and kcat as previously described. Differences between the two enzymes were clearly detected in the activation energies E,1 and E-1, which are significantly higher for cruzipain. The corresponding AS, and AS-1 were positive and significantly higher for cruzipaina(au)|
|Appears in Collections:||Em verificação - Dissertações e teses|
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