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 mo tif. In this study, through systematic site-directed mutagenesis, we have c onfirmed the accompanying biochemical studies and have shown that both CB-1 and CB-2 are involved in calcium binding and enzymatic activity. More spec ifically, we identified critical residues (viz, Asp(210), Asp(212), Gly(213 ), and Thr(216) in CB-1 and Asn(375), Tyr(379), and Glu(381) in CB-2) that are important for calcium binding and heparinase I enzymatic activity. Muta tions in CB-1 resulted in a lower k(cat), but did not change the product pr ofile of heparinase I action on heparin; conversely, mutations in CB-2 not only altered the k(cat) for heparinase I, but also resulted in incomplete d egradation, leading to longer saccharides. Fluorescence competition experim ents 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 hepa rinase 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 hepari nase I activity, suggesting a different role for CB-2 in the enzymatic acti on of heparinase I. These results, taken together with our accompanying stu dy, led us to propose a model for calcium binding to heparinase I that incl udes both CB-1 and CB-2 providing critical interactions, albeit via a diffe rent mechanism. Through binding to CB-1 and/or CB-2, we propose that calciu m may play a role in the catalytic mechanism and/or in the exolytic process ive mechanism of heparin-like glycosaminoglycan depolymerization by heparin ase I.