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1.
Biomacromolecules ; 9(1): 363-8, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18163571

RESUMEN

For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx.


Asunto(s)
Glutatión Peroxidasa/metabolismo , Imitación Molecular , Selenio/metabolismo , Sitios de Unión , Catálisis , Electroforesis en Gel de Poliacrilamida , Especificidad por Sustrato
2.
FEBS J ; 274(15): 3846-54, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17617230

RESUMEN

A 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-beta-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, beta-cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6'-diselenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (Ebselen) in the reduction of H(2)O(2), tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate-binding site should match the size and shape of the substrate and second, incorporation of an imido-group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate-induced mitochondria damage system, and this implies its prospective therapeutic application.


Asunto(s)
Cloro/química , Ciclodextrinas/química , Ciclodextrinas/metabolismo , Glutatión Peroxidasa/metabolismo , Compuestos de Organoselenio/química , Compuestos de Organoselenio/metabolismo , Selenio/química , beta-Ciclodextrinas/química , beta-Ciclodextrinas/metabolismo , Animales , Catálisis , Bovinos , Ciclodextrinas/síntesis química , Cinética , Mitocondrias Cardíacas/metabolismo , Estructura Molecular , Compuestos de Organoselenio/síntesis química , Estrés Oxidativo , beta-Ciclodextrinas/síntesis química
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