Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate
Journal Article
·
· Journal of Biological Chemistry
Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a {beta}-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.
- Research Organization:
- Brookhaven National Laboratory (BNL) National Synchrotron Light Source
- Sponsoring Organization:
- DOE - OFFICE OF SCIENCE
- DOE Contract Number:
- AC02-98CH10886
- OSTI ID:
- 1019669
- Report Number(s):
- BNL--95515-2011-JA
- Journal Information:
- Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 26 Vol. 285; ISSN JBCHA3; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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