Biochemical investigations and mapping of the calcium-binding sites of heparinase I from Flavobacterium heparinum

The heparinases from Flavobacterium heparinum are lyases that specifically cleave heparin-like glycosaminoglycans, Previously, amino acids located in the active site of heparinase I have been identified and mapped. In an effo rt to further understand the mechanism by which heparinase I cleaves its po lymer substrate, we sought to understand the role of calcium, as a necessar y cofactor, in the enzymatic activity of heparinase I, Specifically, we und ertook a series of biochemical and biophysical experiments to answer the qu estion of whether heparinase I binds to calcium and, if so, which regions o f the protein are involved in calcium binding. Using the fluorescent calciu m analog terbium, we found that heparinase I tightly bound divalent and tri valent cations, Furthermore, we established that this interaction was speci fic for ions that closely approximate the ionic radius of calcium. Through the use of the modification reagents N-ethyl-5-phenylisoxazolium-3'-sulfona te (Woodward's reagent K) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, we showed that the interaction between heparinase I and cal cium was essential for proper functioning of the enzyme. Preincubation with either calcium alone or calcium in the presence of heparin was able to pro tect the enzyme from inactivation by these modifying reagents. In addition, through mapping studies of Woodward's reagent K-modified heparinase I, we identified two putative calcium-binding sites, CB-1 (Glu(207)-Ala(219)) and CB-2 (Thr(373)-Arg(384)), in heparinase I that not only are specifically m odified by Woodward's reagent K, leading to loss of enzymatic activity, but also conform to the calcium-coordinating consensus motif.