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, 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.
Cazzulo
J J
JJ
Instituto de Investigaciones Bioquímicas Fundación Campomar, Buenos Aires, Argentina.
Bravo
M
M
Raimondi
A
A
Engström
U
U
Lindeberg
G
G
Hellman
U
U
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
D009842:Oligopeptides; D010446:Peptide Fragments; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases; C063214:cruzipain