Subsite specificity (S3, S2, S1 ', S2 ' and S3 ') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity

Subsite specificity (S3, S2, S1 ', S2 ' and S3 ') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity

Author Hemerly, Jefferson P. Autor UNIFESP Google Scholar
Oliveira, Vitor Autor UNIFESP Google Scholar
Del Nery, Elaine Autor UNIFESP Google Scholar
Morty, Rory E. Google Scholar
Andrews, Norma W. Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Yale Univ
Abstract We characterized the extended substrate binding site of recombinant oligopeptidase B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma brucei (Tb-OP), evaluating the specificity of their S3, S2, S1', S2' and S3' subsites. Five series of internally quenched fluorescent peptides based on the substrate Abz-AGGRGAQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this substrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to be hydrolysed by Tc-OP? and Tb-OP. These two oligopeptidases were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg-Xaa bond. We also demonstrated that Tc-OP? and Tb-OP? have a very specific carboxypeptidase activity for basic amino acids, which depends on the presence of at least of a pair of basic amino acids at the C-terminal end of the substrate. the peptide with triple Arg residues (Abz-AGRRRAQ-EDDnp) was an efficient substrate for Tc-OP? and Tb-OP: the Arg-Ala peptide bond was cleaved first and then two C-terminal Arg residues were successively removed. the S F subsite seems to be an important determinant of the specificity of both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. the presence of these amino acids at P1' resulted in substrates that were hydrolysed with K(m) values in the sub-micromolar range. Taken together, this work supports the view that oligopeptidase B is a specialized protein-processing enzyme with a specific carboxypeptidase activity. Excellent substrates were obtained for Tb-OP? and Tc-OP (Abz-AMRRTISQ-EDDnp and Abz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkably high k(cat) and low K(m) values.
Keywords arginyl hydrolase
Chagas disease
prolyl oligopeptidase
sleeping sickness
Language English
Date 2003-08-01
Published in Biochemical Journal. London: Portland Press Ltd, v. 373, p. 933-939, 2003.
ISSN 0264-6021 (Sherpa/Romeo, impact factor)
Publisher Portland Press Ltd
Extent 933-939
Origin http://dx.doi.org/10.1042/BJ20030342
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000184800300031
URI http://repositorio.unifesp.br/handle/11600/27332

Show full item record




File

File Size Format View

There are no files associated with this item.

This item appears in the following Collection(s)

Search


Browse

Statistics

My Account