Kinetics of parasite cysteine proteinase inactivation by NO-donors

被引:27
作者
Bocedi, A
Gradoni, L
Menegatti, E
Ascenzi, P
机构
[1] Univ Roma Tre, Dipartimento Biol, I-00146 Rome, Italy
[2] Univ Roma Tre, Lab Interdipartimentale Microscopia Elettron, I-00146 Rome, Italy
[3] Univ Aquila, Dipartimento Chim Ingn Chim & Mat, I-67100 Laquila, Italy
[4] Ist Super Sanita, Lab Parassitol, I-00161 Rome, Italy
[5] Univ Ferrara, Dipartimento Sci Farmaceut, I-44100 Ferrara, Italy
关键词
cruzipain; falcipain; Leishmania infantum cysteine proteinase; NO-donors; parasite cysteine proteinase inactivation mechanism;
D O I
10.1016/j.bbrc.2004.01.113
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-lsulphonyl (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 pK(ljg) = 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-alpha-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. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:710 / 718
页数:9
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