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

The Biochemical Journal
E D NeryLuiz Juliano

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 P5-P'5, using a fluorescent quenched solid-phase library assay. Positively charged residues appear to be a general preference in the P5-P3 and the P'5-P'3 positions, while a hydrophobic residue was always required at the P2 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 P2 position. The P1 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 P1 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-G...Continue Reading

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