HYDROLYSIS OF SYNTHETIC PEPTIDES BY CRUZIPAIN, THE MAJOR CYSTEINE PROTEINASE FROM TRYPANOSOMA-CRUZI, PROVIDES EVIDENCE FOR SELF-PROCESSING AND THE POSSIBILITY OF MORE SPECIFIC SUBSTRATES FOR THE ENZYME

Two synthetic peptides, peptide G, with the sequence KEEASSAVVGGPG, co nsisting of the last 10 amino acid residues of the catalytic domain, p lus the first 3 at the C-terminal domain, of cruzipain, and peptide R, with the sequence KEEASSAVVRGPG, were hydrolyzed by the enzyme, as sh own by reversed-phase HPLC. Peptide R was the best substrate, with a V -max/K-m ratio 6-fold higher as compared with peptide G, in good agree ment with previous studies indicating that, in small peptides, cruzipa in prefers R or K at P1. The optimal pH values for hydrolysis of pepti des G and R were 6.8 and 8.0, respectively. A p-nitroanilide derivativ e containing the P1-P3 residues, Z-VVR-pNA, was an excellent substrate for cruzipain, with a K-m value (33 mu M at pH 9.0) lower than that f or Bz-PFR-pNA (66 mu M). These results open the possibility of synthes izing more specific substrates and inhibitors of cruzipain.