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 ε-NH2-Cap-Leu-Xaa-MCA (where Cap is ε-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.
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April 1997
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Research Article|
April 15 1997
Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides
Elaine DEL NERY;
Elaine DEL NERY
*Department of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020 São Paulo, Brazil
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Maria A. JULIANO;
Maria A. JULIANO
*Department of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020 São Paulo, Brazil
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Morten MELDAL;
Morten MELDAL
†Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby-Copenhagen, Denmark
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Ib SVENDSEN;
Ib SVENDSEN
†Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby-Copenhagen, Denmark
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Julio SCHARFSTEIN;
Julio SCHARFSTEIN
‡Molecular Immunology Laboratory, Instituto de Biofisica Carlos Chagas Filho, C. C. S. Universidade Federal do Rio de Janeiro, Rio de Janeiro 21949, Brazil
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Adrian WALMSLEY;
Adrian WALMSLEY
§Department of Molecular Biology and Biotechnology, University of Sheffield, P.O. Box 594, First Court Western Bank, Sheffield S10 2UH, U.K.
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Luiz JULIANO
Luiz JULIANO
‖
*Department of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, 04044-020 São Paulo, Brazil
‖To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
July 10 1996
Revision Received:
December 09 1996
Accepted:
December 12 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 323 (2): 427–433.
Article history
Received:
July 10 1996
Revision Received:
December 09 1996
Accepted:
December 12 1996
Citation
Elaine DEL NERY, Maria A. JULIANO, Morten MELDAL, Ib SVENDSEN, Julio SCHARFSTEIN, Adrian WALMSLEY, Luiz JULIANO; Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides. Biochem J 15 April 1997; 323 (2): 427–433. doi: https://doi.org/10.1042/bj3230427
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