Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationship. Moreover, they appear as promising targets for antiparasite chemotherapy. Here, the first quantitative investigation on the steady-state and pre-steady-state kinetics of the papain-like cysteine proteinases from epimastigotes of Trypanosoma cruzi (cruzipain), the agent of Chagas' disease, and from promastigotes of Leishmania infantum, an agent of visceral and cutaneous leishmaniases, is reported. The results indicate that kinetics for the parasite proteinase catalyzed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalatlyl-L-arginine-7-amino-4-methylcou marin) may be consistently fitted to the minimum three-step mechanism involving the acyl enzyme intermediate ER: [GRAPHICS] At neutral pH, the k(+3) step (deacylation process) is rate limiting in enzyme catalysis, whereas, at pH < 6, the k,., step (acylation process) becomes rate limiting. This illustrates the potential danger in interpreting both k(cad) versus pH profile, given that the acylation or the deacylation step is rate limiting throughout the whole pH range explored, and K-m as the true affinity constant for the E: S complex formation. Comparison with the steady-state and pre-steady-state kinetics of homologous plant enzymes suggests that the parasite cysteine proteinase catalytic behavior appears to be of general significance. (C) 2003 Elsevier Inc. All rights reserved.