title: 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 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-Gln-EDDnp series, whereas the mammalian enzyme was able to tolerate large P1 residues, such as phenylalanine, better than cruzipain in the latter series.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020 São Paulo, Brazil.
authors.forename: E D
authors.initials: ED
authors.lastname: Nery
authors.forename: M A
authors.initials: MA
authors.lastname: Juliano
authors.forename: M
authors.initials: M
authors.lastname: Meldal
authors.forename: I
authors.initials: I
authors.lastname: Svendsen
authors.forename: J
authors.initials: J
authors.lastname: Scharfstein
authors.forename: A
authors.initials: A
authors.lastname: Walmsley
authors.forename: L
authors.initials: L
authors.lastname: Juliano
mesh_terms: D000409:Alanine; D000818:Animals; D000953:Antigens, Protozoan / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D016208:Databases, Factual; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D010455:Peptides / Q000378:metabolism*; D011392:Proline; D015800:Protozoan Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D000953:Antigens, Protozoan; D004792:Enzyme Precursors; D005456:Fluorescent Dyes; D006023:Glycoproteins; D010455:Peptides; D015800:Protozoan Proteins; D011392:Proline; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C063214:cruzipain; D000409:Alanine