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Title: Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides
Authors: Del Nery, Elaine [UNIFESP]
Juliano, Maria Aparecida [UNIFESP]
Meldal, Morten
Svendsen, Ib
Scharfstein, Julio
Walmsley, Adrian
Juliano, Luiz [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
Universidade Federal do Rio de Janeiro (UFRJ)
Issue Date: 15-Apr-1997
Publisher: Portland Press
Citation: Biochemical Journal. London: Portland Press, v. 323, n. 2, p. 427-433, 1997.
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.
ISSN: 0264-6021
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