SUBSTRATE SPECIFICITY OF THE M32 CARBOXYPEPTIDASE OF TRYPANOSOMA BRUCEI.

FRASCH, ALEJANDRA PAULA; NIEMIROWICZ, GABRIELA TERESA; CARMONA, ADRIANA; JULIANO, LUIZ; CAZZULO, JUAN JOSÉ

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protozología

Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms but they are absent from eukaryotic genomes with the remarkable exception of those of trypanosomatids. The genome of T. brucei, the causative agent of Sleeping sickness, encodes one such MCP which displays 72% identity to the characterized TcMCP-1. As its orthologue, T. brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite. Purified recombinant TbMCP-1 exhibits a significant hydrolytic activity against the carboxypeptidase B (subfamily M14B)substrate FA (furylacryloil)-Ala-Lys at pH 7.0-7.8 resembling the T. cruzi enzyme. Several divalent cations had little effect on TbMCP-1 activity but increasing amounts of Co2+ inhibited the enzyme. Despite having similar tertiary structure, both protozoan MCPs display different substrate specificity with respect to P1 position. Thus, TcMCP-1 enzyme cleaved Abz-FVK-(Dnp)-OH substrate (where Abz: o-aminobenzoic acid and Dnp: 2,4-dinitro-phenyl) whereas TbMCP-1 had no activity on this peptide. Comparative homology models and sequence alignments using TcMCP-1 as a template led us to map several residues that could explain this difference. To verify this hypothesis, site-directed mutagenesis was undertaken replacing the TbMCP-1 residues by those present in TcMCP-1. We found that the substitution A414M led TbMCP-1 to gain activity on Abz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination, probably being part of the S1 sub-site. Moreover, the activity of both protozoan MCPs was explored on two vasoactive compounds such as bradikinin (BK) and angiotensinogen (Ang) resulting in two different and distinctive hydrolysis patterns. The biochemical characterization of this peptidase plus the RNAi approach under way, may allow us to validate this enzyme as a chemotherapy target.