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

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

Author Judice, Wagner Alves de Souza Autor UNIFESP Google Scholar
Cezari, Maria Helena Sedenho Autor UNIFESP Google Scholar
Lima, APCA Google Scholar
Scharfstein, J. Google Scholar
Chagas, Jair Ribeiro Autor UNIFESP Google Scholar
Tersariol, Ivarne Luis dos Santos Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Universidade Federal do Rio de Janeiro (UFRJ)
Univ Mogi das Cruzes
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.
Keywords cruzipain
cruzain
cysteine-proteinase
C-terminal domain
substrate specificity
Language English
Date 2001-12-01
Published in European Journal of Biochemistry. Oxford: Blackwell Publishing Ltd, v. 268, n. 24, p. 6578-6586, 2001.
ISSN 0014-2956 (Sherpa/Romeo, impact factor)
Publisher Blackwell Publishing Ltd
Extent 6578-6586
Origin http://dx.doi.org/10.1046/j.0014-2956.2001.02612.x
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000172788400026
URI http://repositorio.unifesp.br/handle/11600/26667

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