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

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.