The calcium-binding sites of heparinase I from Flavobacterium heparinum are essential for enzymatic activity.

In the accompanying paper (Shriver, Z., Liu, D., Hu, Y., and Sasisekharan, R. (1999) J. Biol. Chem. 274, 4082-4088), we have shown that calcium binds specifically to heparinase I and have identified two major calcium-binding sites (CB-1 and CB-2) that partly conform to the EF-hand calcium-binding motif. In this study, through systematic site-directed mutagenesis, we have confirmed the accompanying biochemical studies and have shown that both CB-1 and CB-2 are involved in calcium binding and enzymatic activity. More specifically, we identified critical residues (viz. Asp210, Asp212, Gly213, and Thr216 in CB-1 and Asn375, Tyr379, and Glu381 in CB-2) that are important for calcium binding and heparinase I enzymatic activity. Mutations in CB-1 resulted in a lower kcat, but did not change the product profile of heparinase I action on heparin; conversely, mutations in CB-2 not only altered the kcat for heparinase I, but also resulted in incomplete degradation, leading to longer saccharides. Fluorescence competition experiments along with heparin affinity chromatography suggested that mutations in CB-1 alter heparinase I activity primarily through decreasing the enzyme's affinity for its calcium cofactor without altering heparin binding to heparinase I. Compared with CB-1 mutations, mutations in CB-2 affected calcium binding to a lesser extent, but they had a more pronounced effect on heparinase I activity, suggesting a different role for CB-2 in the enzymatic action of heparinase I. These results, taken together with our accompanying study, led us to propose a model for calcium binding to heparinase I that includes both CB-1 and CB-2 providing critical interactions, albeit via a different mechanism. Through binding to CB-1 and/or CB-2, we propose that calcium may play a role in the catalytic mechanism and/or in the exolytic processive mechanism of heparin-like glycosaminoglycan depolymerization by heparinase I.