Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides

Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides

Author Del Nery, Elaine Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Meldal, Morten Google Scholar
Svendsen, Ib Google Scholar
Scharfstein, Julio Google Scholar
Walmsley, Adrian Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
DEPT CHEM
Universidade Federal do Rio de Janeiro (UFRJ)
UNIV SHEFFIELD
Abstract The substrate specificity of the major cysteinyl proteinase of the parasitic protozoan Trypanosoma cruzi (cruzipain) was investigated, by combinatorial replacement of amino acid residues at positions P-5-P'(5), using a fluorescent quenched solid-phase library assay. Positively charged residues appear to be a general preference in the P-5-P-3 and the P'(5)-P'(3) positions, while a hydrophobic residue was always required at the P-2 position. A broad range of amino acids could be accepted at the P'(1) position. A clear difference in terms of specificity between cruzipain and human cathepsin L was observed for the accommodation of Pro at the P-2 position. The P-1 specificity was investigated by a more detailed enzyme kinetic analysis using peptidyl-MCA (where MCA is methylcoumarin amide) and Abz-peptidyl-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] as substrates, and the results were compared with those obtained using human cathepsin L. Cruzipain showed a clear preference for benzyl-Cys or Arg at the P-1 position. Human cathepsin L presented similar behaviour to that of cruzipain for the hydrolysis of the epsilon-NH2-Cap-Leu-Xaa-MCA (where Cap is epsilon-aminocaproyl) and Abz-Lys-Leu-Xaa-Phe-Ser-Lys-Gln-EDDnp series, whereas the mammalian enzyme was able to tolerate large P-1 residues, such as phenylalanine, better than cruzipain in the latter series.
Language English
Date 1997-04-15
Published in Biochemical Journal. London: Portland Press, v. 323, n. 2, p. 427-433, 1997.
ISSN 0264-6021 (Sherpa/Romeo, impact factor)
Publisher Portland Press
Extent 427-433
Origin http://dx.doi.org/10.1042/bj3230427
Access rights Open access Open Access
Type Article
Web of Science ID WOS:A1997WV76200017
URI http://repositorio.unifesp.br/11600/42800

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