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

• Autor(a) principal: Del Nery, Elaine [UNIFESP]
• Data de Publicação: 1997
• Outros Autores: Juliano, Maria Aparecida [UNIFESP], Meldal, Morten, Svendsen, Ib, Scharfstein, Julio, Walmsley, Adrian, Juliano, Luiz [UNIFESP]
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UNIFESP
• dARK ID: ark:/48912/0013000008hbv
• DOI: 10.1042/bj3230427
• Texto Completo: http://dx.doi.org/10.1042/bj3230427; http://repositorio.unifesp.br/handle/11600/42800

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.