PRELIMINARY CHARACTERIZATION OF A NEW METALLOCARBOXYPEPTIDASE OF T. CRUZI.

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

Bariloche

Congreso; BARILOCHE PROTEIN SYMPOSIUM; 2003

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular, Sociedad Argentina de Biofísica.

A search on unassembled shotgun reads derived from T. cruzi Genome Project reveled two open reading frames with homology to metallocarboxypeptidases of the M32 family. The ORFs had 1512pb and 1506pb, and predicted two proteins 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 archaea and bacteria. Homologous genes in eukaryotic organisms have been detected only in trypanosomatids (L. major and now T. cruzi) to date. This fact may offer a new possible target for the chemotherapy of Chagas disease. We designed specific primers to amplify TcCP-1 gen by PCR and expressed it in E. coli cells, as an NH2-terminal His6-tagged protein. Purification of TcCP-1 with an IMAC column charged with Co+2 gave an active protein with a molecular mass of 61.6 KDa (by MALDI-ToF MS) which was inhibited by 1mM EDTA, thus showing its metallopeptidase nature. Despite this fact, metal ions such as Ni+2, Zn+2, and Co+2 to a lower extent, also inhibited the recombinant enzyme. TcCP-1 presented a pH optimum around 6, when acting on the N-blocked synthetic dipeptides furylacryloyl(FA)-Ala-Lys and FA-Phe-Phe. Preliminary kinetic studies were not consistent with a simple Michaelis-Menten kinetics, showing a biphasic behavior and positive cooperativity (n=1,74). This is the first enzyme of this family to be reported in a eukariotic organism.