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 h eparinase I (EC 4.2.2.7). Chemical and proteolytic digests of heparina se I were used in direct binding and competition assays, to map the re gions of heparinase I that interact specifically with heparin. We find the heparin binding site contains two Cardin-Weintraub heparin bindin g consensus sequences and a calcium co-ordination consensus motif. We show that heparin binding to heparinase I is independent of calcium (K -d of 60 nM) and that calcium is able to activate heparinase I catalyt ically. 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 b inding site to the active site. Site-directed mutagenesis of H203A (co ntained in the heparin binding site) inactivated heparinase I; however , a H203D mutant retained marginal activity, indicating a role for thi s 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 ac tive site are in close proximity to each other and that the calcium co ordination motif, contained in the heparin binding site, may bridge he parin to heparinase I through calcium in a ternary complex during cata lysis.