Please use this identifier to cite or link to this item: http://repositorio.unifesp.br/handle/11600/26667
Title: Comparison of the specificity, stability and individual rate constants with respective activation parameters for the peptidase activity of cruzipain and its recombinant form, cruzain, from Trypanosoma cruzi
Authors: Judice, Wagner Alves de Souza [UNIFESP]
Cezari, Maria Helena Sedenho [UNIFESP]
Lima, APCA
Scharfstein, J.
Chagas, Jair Ribeiro [UNIFESP]
Tersariol, Ivarne Luis dos Santos [UNIFESP]
Juliano, Maria Aparecida [UNIFESP]
Juliano, Luiz [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
Universidade Federal do Rio de Janeiro (UFRJ)
Univ Mogi das Cruzes
Keywords: cruzipain
cruzain
cysteine-proteinase
C-terminal domain
substrate specificity
Issue Date: 1-Dec-2001
Publisher: Blackwell Publishing Ltd
Citation: European Journal of Biochemistry. Oxford: Blackwell Publishing Ltd, v. 268, n. 24, p. 6578-6586, 2001.
Abstract: The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid 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 mannose-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. S-2 to S-2' subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, K-m 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 k(cat) value 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 (k(1), substrate diffusion; k(-1), substrate dissociation; k(2), acylation; k(3), deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis-Menten parameters k(cat)/K-m and k(cat) as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci. 9, 1589-1593]. 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 DeltaS(1) and DeltaS(-1) were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain.
URI: http://repositorio.unifesp.br/handle/11600/26667
ISSN: 0014-2956
Other Identifiers: http://dx.doi.org/10.1046/j.0014-2956.2001.02612.x
Appears in Collections:Em verificação - Geral

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