Catalytic mechanism of Heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate

Download
  1. (PDF, 1 MB)
  2. Get@NRC: Catalytic mechanism of Heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1074/jbc.M110.101071
AuthorSearch for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for:
TypeArticle
Journal titleJournal of biological chemistry
Volume285
Issue26
Pages2005120061; # of pages: 11
AbstractHeparinase 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.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Biotechnology Research Institute
Peer reviewedYes
NRC number50698
NPARC number16512458
Export citationExport as RIS
Report a correctionReport a correction
Record identifier45825d8d-4984-4524-b567-ccba7fc440f0
Record created2010-12-14
Record modified2016-05-09
Bookmark and share
  • Share this page with Facebook (Opens in a new window)
  • Share this page with Twitter (Opens in a new window)
  • Share this page with Google+ (Opens in a new window)
  • Share this page with Delicious (Opens in a new window)