Comparative substrate specificity of M32 metallocarboxypeptidases in trypanosomatids

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

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular.; 2012

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

P { margin-bottom: 0.21cm; } 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 exception of those of trypanosomatids. The genome of Trypanosoma brucei, the causative agent of Sleeping Sickness, encodes one MCP which displays 72% identity to the characterized TcMCP-1 from T. cruzi. As its orthologue, T. brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite but it displays a different substrate specificity with respect to P1 position. To further explore the enzyme specificity, we employed 4 positional-scanning synthetic combinatorial libraries of fluorescence resonance energy transfer peptides. These peptides, with general sequences AbzGXXRZK(Dnp)OH; AbzGXXZXK(Dnp)OH; AbzGXZRXK(Dnp)OH; and AbzGZXRXK(Dnp)OH (where Z was successively occupied with one of the 19 amino acids with the exception of Cys and X is a random residue), contain the ortho-aminobenzoyl (Abz) and 2,4-dinitrophenyl (Dnp) as a donor/acceptor pair and permitted to study the P1, P2, P3, P4 substrate preference of recombinant MCPs. Our results indicate that TbMCP-1 had a more restricted selectivity for Phe in P1 position compared to TcMCP-1, which presented a wide range of substrate utilization. On the other hand, the S2, S3 and S4 subsites of both MCPs could accommodate a broad range of residues.