Kinetics of parasite cysteine proteinase inactivation by NO-donors

Abstract

NO-donors block Plasmodium, Trypanosoma, and Leishmania life cycle inactivating parasite cysteine proteinases. In this study, the inactivation of falcipain, cruzipain, and Leishmania infantum cysteine proteinase by S-nitroso-5-dimethylaminonaphthalene-1-sulphonyl (dansyl-SNO), S-nitrosoglutathione (GSNO), (±)-( E)-4-ethyl-2-[( E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), and S-nitrosoacetylpenicillamine (SNAP) is reported. With NO-donors in excess over the parasite cysteine proteinase, the time course of enzyme inactivation 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 NO-donor concentrations but tends to first-order at high NO-donor concentrations. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting pseudo-first-order process. Kinetic parameters of cruzipain inactivation by GSNO were affected by the acidic p K shift of one ionizing group (from p K unl=5.7 to p K lig=4.8) upon GSNO-induced enzyme inactivation. Falcipain, cruzipain, and L. infantum cysteine proteinase inactivation by dansyl-SNO, GSNO, NOR-3, and SNAP is prevented and reversed by dithionite and l-ascorbic acid. However, the incubation of L. infantum cysteine proteinase with dansyl-SNO does not result in the appearance of fluorescence of the enzyme. More than 90% of the S-transnitrosylation product GSH existed in the inactivation reaction, suggesting that S-transnitrosylation is the favorite process for parasite cysteine proteinase inactivation. Furthermore, the fluorogenic substrate N-α-benzyloxycarbonyl- l-phenylalanyl- l-arginine-(7-amino-4-methylcoumarin) protects L. infantum cysteine proteinase from inactivation by SNAP. These results indicate that parasite cysteine proteinase inactivation by NO-donors occurs via NO-mediated S-nitrosylation of the Cys25 catalytic residue.