title: Analysis of peptidase activities of a cathepsin B-like (TcoCBc1) from Trypanosoma congolense.
abstract: The substrate specificity of TcoCBc1 was evaluated using two internally quenched fluorescent peptide libraries with randomized sequences designed to detect carboxydipeptidase (Abz-GXXZXK(Dnp)-OH) and endopeptidase (Abz-GXXZXXQ-EDDnp) activities at acidic and neutral pHs, respectively. All the data obtained with TcoCBc1 were compared with those of human cathepsin B, including the pH profiles of the hydrolytic reactions. The most relevant observation is the preference of TcoCBc1 for substrates with a pair of acidic amino acids at positions P(2) and P(1) for its carboxydipeptidase activity and the well acceptance for E and D at P(1) position for endopeptidase activity. These peculiar preferences for negatively charged groups of TcoCBc1 and its requirements for carboxydipeptidase activity were also observed on Abz labeled analogues of bradykinin (Abz-RPPG(↓)FSAFR-OH, Abz-RPPG(↓)FS(↓)AF-OH, Abz-RPPG(↓)DE(↓)AF-OH) and angiotensin I (Abz-DR(↓)VYIHAFHL-OH), where (↓) indicates the cleavage site. TcoCBc1 was modeled based on the atomic coordinates of the cathepsin B from Trypanosoma brucei and the positively charged environment in TcoCBc1 catalytic site contrasts with the negatively charged environment in human cathepsin B. The preferences of S1 and S2 subsites of TcoCBc1 for acidic amino acids have to be taken into consideration for future studies of physiological roles of TcoCBc1 as for instance in apoptotic processes of Trypanosoma congolense.
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Lilian C G
authors.initials: LC
authors.lastname: Oliveira
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Débora N
authors.initials: DN
authors.lastname: Okamoto
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Juliana R
authors.initials: JR
authors.lastname: Oliveira
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Marcia Y
authors.initials: MY
authors.lastname: Kondo
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Iuri E
authors.initials: IE
authors.lastname: Gouvea
authors.affiliation: CNRS, Université Bordeaux Segalen, Microbiologie fondamentale et Pathogénicité, UMR 5234, F-33000 Bordeaux, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Biteau
authors.affiliation: CNRS, Université Bordeaux Segalen, Microbiologie fondamentale et Pathogénicité, UMR 5234, F-33000 Bordeaux, France.
authors.forename: Theo
authors.initials: T
authors.lastname: Baltz
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisas em Energia e Materiais, Giuseppe Maximo Scolfaro 10000, Campinas 13083-970, Brazil.
authors.forename: Mário T
authors.initials: MT
authors.lastname: Murakami
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil. Electronic address: map.juliano@gmail.com.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
mesh_terms: D000803:Angiotensin I / Q000378:metabolism*; D001920:Bradykinin / Q000378:metabolism*; D020134:Catalytic Domain; D002401:Cathepsin B / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism*; D019151:Peptide Library; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014348:Trypanosoma congolense / Q000201:enzymology*
chemical_list: D010446:Peptide Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D000803:Angiotensin I; D002401:Cathepsin B; D001920:Bradykinin
keywords: Carboxydipeptidase; Endopeptidase; Fluorescent substrate; Peptide library; Protease