BIOCHEMICAL CHARACTERIZATION OF A METALLOCARBOXYPEPTIDASE OF Trypanosoma brucei.

FRASCH AP; NIEMIROWICZ G; CAZZULO JJ

San Miguel de Tucumán, Tucumán, Argentina.

Congreso; XLV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.

Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms. Members of this family are absent from eucaryotic 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, the T. brucei enzyme is cytosolic andacts best on Furyl-Acryloyl-AK at pH 7.3 with a Km of 260 μM. Divalent cations such as Ni2+ and Zn2+ had little effect on TbMCP-1 activity., but increasing amounts of Co2+ inhibited the enzyme. Despite having similar tertiary structure, both MCPs display different substrate specificity with respect to P1 position. Whereas TcMCP-1 cleaves Abz-FVK-(Dnp)-OH (where Abz: oaminobenzoic acid and Dnp: 2,4-dinitrophenyl) TbMCP-1 had noactivity on this substrate. Comparative homology models and sequence alignments using TcMCP-1 as template, led us to map several residues that could explain this difference. To examine the role that these residues could play in P1 preference, site-directed mutagenesis was undertaken replacing the TbMCP-1 specific residues by those present in TcMCP-1. We found that the substitution of Ala414 by Met414 led TbMCP-1 to gain activity onAbz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination.