Cysteine proteases are relevant to several aspects of the parasite life cycle and the parasite-host relationship. Moreover, they appear as promising targets for antiparasite chemotherapy. Here, a quantitative investigation on the catalytic properties of cruzain, the papain-like cysteine protease from epimastigotes of Trypanosoma cruzi, is reported. The results indicate that kinetics for the cruzain catalyzed hydrolysis of N-α-benzyloxycarbonyl-L- arginyl-L-alanine-(7-amino-4-methylcoumarin), N-α-benzyloxycarbonyl-L- phenylalanyl-L-alanine-(7-amino-4-methylcoumarin), and N-α- benzyloxycarbonyl-L-tyrosyl-L-alanine-(7-amino-4-methylcoumarin) can be consistently fitted to the minimum three-step mechanism of cysteine proteases involving the acyl·enzyme intermediate E·P; the deacylation step is rate-limiting in enzyme catalysis. Remarkably, these substrates show identical catalytic parameters. This reflects the ability of the cruzain Glu205 residue, located at the bottom of the S 2 subsite, to neutralize the substrate/inhibitor polar P 2 residues (e.g., Arg or Tyr) and to be solvent-exposed when substrate/inhibitor nonpolar P 2 residues (e.g., Phe) fit the S 2 subsite. More complex catalytic mechanisms are also discussed. Binding free-energy calculation provides a quantitative framework for the interpretation of these results; in particular, direct evidence for the compensatory effect between Coulomb interaction(s) and solvation effect(s) is reported. These results appear of general significance for a deeper understanding of (macro)molecular recognition and for the rational design of novel inhibitors of parasitic cysteine proteases.
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