HEPARINASE-I FROM FLAVOBACTERIUM-HEPARINUM - IDENTIFICATION OF A CRITICAL HISTIDINE RESIDUE ESSENTIAL FOR CATALYSIS AS PROBED BY CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS

We recently identified cysteine-135 as an important amino acid for hep arinase I (EC 4.2.2.7) activity. In this study, we have identified a s econd residue critical for enzymatic activity. We observe concentratio n-dependent inactivation of heparinase I in the presence of reversible histidine-modifying diethyl pyrocarbonate (DEPC); 0.3 mM DEPC results in 95% of heparinase I inactivation in less than 3 min, and as low as 10 mu M DEPC results in a 85% loss of heparinase I activity in 15 min . Heparinase I activity is restored following hydroxylamine treatment. This, along with other experiments, strongly suggests that the inacti vation of heparinase I by DEPC is specific for histidine residues. Che mical modification, under nondenaturing conditions, of the histidines using nonradiolabeled and [C-14]DEPC indicates that between one and tw o histidine residues are modified. Chemical modification of the surfac e-accessible histidines, in the presence and absence of heparin, sugge sts that the histidine(s) lie(s) in or near the active site of heparin ase I. The wild-type heparinase I has four histidine residues; site-di rected mutagenesis of H129A, H165A, and H339A did not affect enzyme ac tivity and the kinetic parameters, suggesting that these residues are not essential for heparinase I activity. However, H203A inactivates he parinase I while a H203D mutant has residual activity, indicating a ro le of this residue in catalysis. We propose that histidine-203, contai ned in the heparin binding site, is immediately adjacent to cysteine-1 35, and these residues together form the catalytic domain of heparinas e I.