Heparinases are bacterial enzymes that are powerful tools to study the physiological roles of heparin-like complex polysaccharides. In addition, heparinases have significant therapeutic applications. We had proposed earlier that cysteine 135 and histidine 203 together form the catalytic domain in heparinase I. We had also identified a heparin binding domain in heparinase I containing two positively charged clusters HB-1 and HB-2 in a primary heparin binding site and other positively charged residues in the vicinity of cysteine 135. In this study, through systematic site-directed mutagenesis studies, we show that the alteration of the positive charge of the HB-1 region has a pronounced effect on heparinase I activity. More specifically, site-directed mutagenesis of K199A (contained in HB-1) results in a 15-fold reduction in catalytic activity, whereas a K198A mutation (also in HB-1) results in only a 2-to 3-fold reduction in heparinase I activity. A K132A mutation, in close proximity to cysteine 135, also resulted in reduced (8-fold) activity. Heparin affinity chromatography experiments indicated moderately lowered binding affinities for the K132A, K198A, and the K199A mutant enzymes. The above results, taken together with our previous observations, lead us to propose that the positively charged heparin binding domain provides the necessary microenvironment for the catalytic domain of heparinase I. The dominant effect of lysine 199 suggests an additional, more direct, role in catalysis for this residue.
Heparin-like glycosaminoglycans (HLGAGs)1 play an intricate role in the extracellular matrix, regulating a wide variety of biological functions (1, 2). HLGAGs are highly sulfated, complex, acidic polysaccharides consisting of alternating uronic acid (L-iduronic or D-glucuronic acid) and D-glucosamine residues connected through 1-4 linkages. Variations in the degree and distribution of sulfation result in a high degree of chemical heterogeneity in HLGAGs. Three enzymes that degrade HLGAGs (heparin and heparan sulfate), viz. heparinases I, II, and III from Flavobacterium heparinum, recognize unique sequences of sulfation and uronic acid epimerization in HLGAGs with a high degree of specificity (3-5). Heparinases have important clinical applications such as in the monitoring of heparin levels in blood (approved by the FDA) (6), neutralization of heparin in blood (in phase III clinical trials), and in production of low molecular weight heparins for use in humans. In addition, heparinases I and III are potent inhibitors of neovascularization (7). More importantly, heparinases have proven to be useful tools in understanding the important physiological roles of HLGAGs (8, 9).Our group has cloned, sequenced, and expressed in Escherichia coli the genes for heparinases I, II, and III from F. heparinum (10 -13). We subsequently carried out extensive biochemical studies to investigate structure-activity relationships of heparinase I and to understand the mechanism of heparin degradation (14 -16). Earlier we showed that cystei...