PRELIMINARY CHARACTERIZATION OF A NEW METALLOCARBOXYPEPTIDASE OF T. CRUZI.

GABRIELA NIEMIROWICZ; FABIOLA PARUSSINI; FERNÁN AGÜERO; DANIEL SÁNCHEZ; JUAN JOSÉ CAZZULO

Bariloche

Congreso; XXXIX Annual Meeting SAIB, XXXII Annual Meeting SAB, Bariloche Protein Symposium; 2003

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

A search on unassembled shotgunreads derived from T. cruzi Genome Project revealed twoopen reading frames with homology to metallocarboxypeptidases of theM32 family. The ORFs had 1512pb and 1506pb, and predicted twoproteins with 64% of identity between them: TcCP-1and TcCP-2.These enzymes belong to a new family of peptidaseswhose members had been found so far exclusively in archaeaand bacteria. Homologous genes in eukaryotic organisms have beendetected only in trypanosomatids (L. major and now T.cruzi) to date. This fact may offer a new possible target forthe chemotherapy of Chagas disease. We designed specificprimers to amplify TcCP-1 gen by PCR and expressed it in E. colicells,as an NH2-terminal His6-tagged protein. Purification ofTcCP-1 with an IMAC column charged with Co+2 gave an active protein with amolecular mass of 61.6 KDa (by MALDI-ToF MS) which wasinhibited by 1mM EDTA, thus showing itsmetallopeptidase nature. Despite this fact, metal ions such as Ni+2, Zn+2, and Co+2to a lower extent, also inhibited the recombinant enzyme.TcCP-1 presented a pH optimum around 6, when acting on theN-blocked synthetic dipeptides furylacryloyl(FA)-Ala-Lysand FA-Phe-Phe. Preliminary kinetic studies were not consistentwith a simple Michaelis-Menten kinetics, showing a biphasicbehavior and positive cooperativity (n=1,74). This is the first enzyme ofthis family to be reported in a eukariotic organism.