NO-donors block Plasmodium, Trypanosoma, and Leishmania life cycle by inactivating parasite enzymes, e.g., cysteine proteinases. In this study, the inactivation of falcipain, cruzipain, and Leishmania infantum cysteine proteinase by the NO-donor 4-(phenylsulfonyl)-3-((2-(dimethylamino)ethyl)thio)-furoxan oxalate (SNO-102) is reported. SNO-102 inactivates dose- and time-dependently parasite cysteine proteinases; one equivalent of NO, released from SNO-102, inactivates one equivalent of L. infantum cysteine proteinase. With SNO-102 in excess over the parasite cysteine proteinase, the time course of enzyme inhibition corresponds to a pseudo-first-order reaction for more than 90% of its course. The concentration dependence of the pseudo-first-order rate constant is second-order at low SNO-102 concentration but tends to first-order at high NO-donor concentration. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting pseudo-first order process. Kinetic parameters of L. infantum cysteine proteinase inactivation by SNO-102 are affected by the acidic pK shift of one apparent ionizing group (from pK(unl)=5.8 to pK(lig)=4.7) upon enzyme inhibition. Falcipain, cruzipain and L. infantum cysteine proteinase inactivation is prevented and reversed by dithiothreitol and L-ascorbic acid. Furthermore, the fluorogenic substrate N-alpha-benzyloxycarbonyl-Phe-Arg-(7-amino-4-methylcoumarin) protects parasite cysteine proteinases from inactivation by SNO-102. The absorption spectrum of the inactive S-nitrosylated SNO-102-treated L. infantum cysteine proteinase displays a maximum at about 340 nm. These results indicate that the parasite cysteine proteinase inactivation by SNO-102 occurs via the NO-mediated S-nitrosylation of the Cys25 catalytic residue.