title: 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.
abstract: Two synthetic peptides, peptide G, with the sequence KEEASSAVVGGPG, consisting of the last 10 amino acid residues of the catalytic domain, plus the first 3 at the C-terminal domain, of cruzipain, and peptide R, with the sequence KEEASSAVVRGPG, were hydrolyzed by the enzyme, as shown by reversed-phase HPLC. Peptide R was the best substrate, with a Vmax/K(m) ratio 6-fold higher as compared with peptide G, in good agreement with previous studies indicating that, in small peptides, cruzipain prefers R or K at P1. The optimal pH values for hydrolysis of peptides G and R were 6.8 and 8.0, respectively. A p-nitroanilide derivative containing the P1-P3 residues, Z-VVR-pNA, was an excellent substrate for cruzipain, with a K(m) value (33 microM at pH 9.0) lower than that for Bz-PFR-pNA (66 microM). These results open the possibility of synthesizing more specific substrates and inhibitors of cruzipain.
authors.affiliation: Instituto de Investigaciones Bioquímicas Fundación Campomar, Buenos Aires, Argentina.
authors.forename: J J
authors.initials: JJ
authors.lastname: Cazzulo
authors.forename: M
authors.initials: M
authors.lastname: Bravo
authors.forename: A
authors.initials: A
authors.lastname: Raimondi
authors.forename: U
authors.initials: U
authors.lastname: Engström
authors.forename: G
authors.initials: G
authors.lastname: Lindeberg
authors.forename: U
authors.initials: U
authors.lastname: Hellman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
chemical_list: D009842:Oligopeptides; D010446:Peptide Fragments; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases; C063214:cruzipain