| Download | - View accepted manuscript: Catalytic mechanism of Heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate (PDF, 1.3 MiB)
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| DOI | Resolve DOI: https://doi.org/10.1074/jbc.M110.101071 |
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| Author | Search for: Shaya, David; Search for: Zhao, Wenjing; Search for: Garron, Marie-Lise; Search for: Xiao, Zhongping; Search for: Cui, Qizhi1; Search for: Zhang, Zhenqing; Search for: Sulea, Traian1; Search for: Linhardt, Robert J.; Search for: Cygler, Miroslaw1 |
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| Affiliation | - National Research Council of Canada. NRC Biotechnology Research Institute
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| Format | Text, Article |
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| Abstract | 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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| Publication date | 2010-06-25 |
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| Publisher | American Society for Biochemistry and Molecular Biology |
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| Language | English |
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| Peer reviewed | Yes |
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| NRC number | NRCC 50698 |
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| NPARC number | 16512458 |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | 45825d8d-4984-4524-b567-ccba7fc440f0 |
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| Record created | 2010-12-14 |
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| Record modified | 2020-04-17 |
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