Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides.
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.
Nery
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ED
Department of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020 São Paulo, Brazil.
Juliano
M A
MA
Meldal
M
M
Svendsen
I
I
Scharfstein
J
J
Walmsley
A
A
Juliano
L
L
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*
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