Catalytic properties of cysteine proteinases from Trypanosoma cruzi and Leishmania infantum: a pre-steady-state and steady-state study.
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-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) may be consistently fitted to the minimum three-step mechanism involving the acyl.enzyme intermediate E.P: [mechanism: see text] At neutral pH, the k(+3) step (deacylation process) is rate limiting in enzyme catalysis, whereas, at pH<6, the k(+2) step (acylation process) becomes rate limiting. This illustrates the potential danger in interpreting both kcat versus pH profile, given that the acylation or the deacylation step is rate limiting throughout the whole pH range explored, and Km 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.
Ascenzi
Paolo
P
Dipartimento di Biologia, Università Roma Tre, Viale Guglielmo Marconi 446, I-00146 Rome, Italy. ascenzi@bio.uniroma3.it
Bocedi
Alessio
A
Visca
Paolo
P
Antonini
Giovanni
G
Gradoni
Luigi
L
D000818:Animals; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D018314:Leishmania infantum / Q000201:enzymology*; D008956:Models, Chemical; D015800:Protozoan Proteins; D014349:Trypanosoma cruzi / Q000201:enzymology*
D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases; C063214:cruzipain