METALLOCARBOXYPEPTIDASES (MCPs) OF Trypanosoma cruzi: EXPRESSION AND CHARACTERIZATION

GABRIELA NIEMIROWICZ, FABIOLA PARUSSINI, FERNÁN AGÜERO, AND JUAN J. CAZZULO

Pinamar, Argentina.

Congreso; PAMBPABMB 10th Congress -Panamerican Association for Biochemistry and Molecular Biology; 2005

Panamerican Association for Biochemistry and Molecular Biology-Sociedad Argentina de Investigación Bioquímica y Biología Molecular-Sociedad Argentina de Neuroquímica.

The genome of Trypanosoma cruzi, the causative agent of Chagas Disease, encodes two MCPs of the M32 family, with 64% of identity between them: TcCP-1 and TcCP-2. These enzymes belong to a new family of peptidases whose members had been found so far exclusively in prokaryotes. This makes them possible candidates as targets for chemotherapy. Both full length TcCP-1 and TcCP-2 genes were expressed in E. coli as catalytically active polyhistidine tagged recombinant enzymes. Despite their homology, purified TcCPs displayed marked differences. TcCP-1 acted optimally at pH 6,2 on furylacryloyl(FA)-Ala-Lys with a Km of 166 μM. Activity against N-carbobenzoxy-Ala-X (ZAX) substrates reveled a P1´ preference for basic and some neutral C-terminal residues. Contrary, TcCP-2 preferred aromatic and alifatic residues at this position. The Km value for FA-Phe-Phe at pH 7,6 was 24 μM. Therefore, the specificity of both MCPs are complementary. Western blot analysis also revealed a different pattern of expression for both enzymes. Whereas TcCP-1 is present in all life cycle stages of T. cruzi, TcCP-2 is mainly expressed in the insect vector ones. Indirect immunofluorescence staining suggested that both proteins are localized in the parasite cytosol.