Kinetics of parasite cysteine proteinase inactivation by NO-donors

Alessio Bocedi, Luigi Gradoni, Enea Menegatti, Paolo Ascenzi

Research output: Contribution to journalArticlepeer-review


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 pK shift of one ionizing group (from pK unl = 5.7 to pKlig = 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.

Original languageEnglish
Pages (from-to)710-718
Number of pages9
JournalBiochemical and Biophysical Research Communications
Issue number3
Publication statusPublished - Mar 12 2004


  • Cruzipain
  • Falcipain
  • Leishmania infantum cysteine proteinase
  • NO-donors
  • Parasite cysteine proteinase inactivation mechanism

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology


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