Probing the cruzain S2 recognition subsite: a kinetic and binding energy calculation study.
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-alpha-benzyloxycarbonyl-l-arginyl-l-alanine-(7-amino-4-methylcoumarin), N-alpha-benzyloxycarbonyl-l-phenylalanyl-l-alanine-(7-amino-4-methylcoumarin), and N-alpha-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.
Polticelli
Fabio
F
Dipartimento di Biologia and Laboratorio Interdipartimentale per la Microscopia Elettronica, Università Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy. polticel@uniroma3.it
Zaini
Germano
G
Bolli
Alessandro
A
Antonini
Giovanni
G
Gradoni
Luigi
L
Ascenzi
Paolo
P
D000818:Animals; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D015195:Drug Design; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D014344:Trypanocidal Agents; D014349:Trypanosoma cruzi / Q000201:enzymology
D015800:Protozoan Proteins; D014344:Trypanocidal Agents; D003546:Cysteine Endopeptidases; C074032:cruzain, Trypanosoma cruzi