HEPARINASE-I FROM FLAVOBACTERIUM-HEPARINUM - THE ROLE OF THE CYSTEINERESIDUE IN CATALYSIS AS PROBED BY CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS

Heparinase I (heparin lyase I, EC 4.2.2.7), a heparin-degrading enzyme produced by Flavobacterium heparinum, is used to deheparinize blood f ollowing extracorporeal procedures in surgery and in other application s. The present study of mapping and characterization of the cysteines of heparinase I represents the first structural characterization of a heparinase. [H-3]Iodoacetic acid labeling demonstrated that heparinase I has two free cysteines. One of the two cysteines is surface accessi ble and lies in a hydrophilic environment while the other is in a hydr ophobic environment. Chemical modification of the cysteines, both in t he presence and in the absence of heparin, suggests that the surface-a ccessible cysteine lies in or near the active site of heparinase I. Pr eferential reactivity of this cysteine with negatively charged sulfhyd ryl-modifying reagents and the cysteines' high reactivity to iodoaceti c acid at pH 6.5 indicate that the surface-accessible cysteine is in a positively charged region. The surface-accessible cysteine (cysteine- 135) was mapped as the active-site cysteine by radiolabeling with [H-3 ]iodoacetic acid and by tryptic digestion and peptide sequencing. Site -directed mutagenesis of cysteine-135 to a serine or an alanine in r-h eparinase I demonstrates that this cysteine is essential for enzymatic activity. However, replacement of the surface-inaccessible cysteine b y a serine or alanine has no effect.