Heparinase I from Flavobacterium heparinum. Mapping and characterization of the heparin binding domain.
In this study we have identified the primary heparin binding site of heparinase I (EC 4.2.2.7). Chemical and proteolytic digests of heparinase I were used in direct binding and competition assays, to map the regions of heparinase I that interact specifically with heparin. We find the heparin binding site contains two Cardin-Weintraub heparin binding consensus sequences and a calcium co-ordination consensus motif. We show that heparin binding to heparinase I is independent of calcium (Kd of 60 nm) and that calcium is able to activate heparinase I catalytically. We find that sulfhydryl selective labeling of cysteine 135 of heparinase I protects the lysines of the heparin binding sequence from proteolytic cleavage, suggesting the close proximity of the heparin binding site to the active site. Site-directed mutagenesis of H203A (contained in the heparin binding site) inactivated heparinase I; however, a H203D mutant retained marginal activity, indicating a role for this residue in catalysis. The above results taken together suggest that histidine 203 (hence the heparin binding site) is immediately adjacent to the scissile bond. We propose that the heparin binding site and active site are in close proximity to each other and that the calcium coordination motif, contained in the heparin binding site, may bridge heparin to heparinase I through calcium in a ternary complex during catalysis.
Sasisekharan
R
R
Harvard-MIT Division of Health Sciences and Technology, and the Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Venkataraman
G
G
Godavarti
R
R
Ernst
S
S
Cooney
C L
CL
Langer
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003488:Cyanogen Bromide; D017931:DNA Primers; D005417:Flavobacterium / Q000201:enzymology*; D006493:Heparin / Q000378:metabolism*; D019763:Heparin Lyase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006493:Heparin; D014357:Trypsin; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase; D003488:Cyanogen Bromide