CHANGING THE SUBSTRATE SPECIFICITIES OF M32 PEPTIDASES OF BY SITE-DIRECT MUTAGENESIS.

GABRIELA NIEMIROWICZ Y JUAN J. CAZZULO

Mar del Plata

Congreso; XLIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Trypanosoma cruzi, the causative agent of Chagas Disease, encodes two metallocarboxypeptidases ( MCPs) of the M32 family which are 64% identical at amino acid sequence level. Despite having very similar tertiary structures, MCPs substrate specificity differ markedly. Whereas MCP-1 cleaves in vitro the C-terminal Arg or Lys residue from peptides and synthetic substrates, MCP-2 prefers aromatic and aliphatic residues at P1´ position. Sequence alignments and homology models, based on the crystal structure of carboxypeptidase, led us to map four sites (Met/Arg-304, Gln/Met-305, Asp/Thr-350, Ala/Ser-351; MCP-1 residues listed first) in MCPs substrate binding channels that appeared to be positioned to account for the differences in specificity. To examine the role that these residues might play in determining P1´ preference, site-directed mutagenesis was undertaken replacing the MCP-1 specific residues by those present in MCP-2. The substitution of Met304, Gln305 by Arg304, Met305 swapped the activity of MCP-1 from furylacryloyl(FA)-Ala-Lys dipeptide towards FA-Phe-Phe substrate, thus showing that those residues are indeed involved in specificity determination.