| Téléchargement | - Voir le manuscrit accepté : Catalytic mechanism of Heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate (PDF, 1.3 Mio)
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| DOI | Trouver le DOI : https://doi.org/10.1074/jbc.M110.101071 |
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| Auteur | Rechercher : Shaya, David; Rechercher : Zhao, Wenjing; Rechercher : Garron, Marie-Lise; Rechercher : Xiao, Zhongping; Rechercher : Cui, Qizhi1; Rechercher : Zhang, Zhenqing; Rechercher : Sulea, Traian1; Rechercher : Linhardt, Robert J.; Rechercher : Cygler, Miroslaw1 |
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| Affiliation | - Conseil national de recherches du Canada. Institut de recherche en biotechnologie du CNRC
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| Format | Texte, Article |
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| Résumé | Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a β-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles. |
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| Date de publication | 2010-06-25 |
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| Maison d’édition | American Society for Biochemistry and Molecular Biology |
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| Dans | |
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| Langue | anglais |
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| Publications évaluées par des pairs | Oui |
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| Numéro du CNRC | NRCC 50698 |
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| Numéro NPARC | 16512458 |
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| Exporter la notice | Exporter en format RIS |
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| Signaler une correction | Signaler une correction (s'ouvre dans un nouvel onglet) |
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| Identificateur de l’enregistrement | 45825d8d-4984-4524-b567-ccba7fc440f0 |
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| Enregistrement créé | 2010-12-14 |
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| Enregistrement modifié | 2020-04-17 |
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