Histidine 295 and Histidine 510 Are Crucial for the Enzymatic Degradation of Heparan Sulfate by Heparinase III

Abstract

The heparinases from Flavobacterium heparinum are powerful tools in understanding how heparin-like glycosaminoglycans function biologically. Heparinase III is the unique member of the heparinase family of heparin-degrading lyases that recognizes the ubiquitous cell-surface heparan sulfate proteoglycans as its primary substrate. Given that both heparinase I and heparinase II contain catalytically critical histidines, we examined the role of histidine in heparinase III. Through a series of diethyl pyrocarbonate modification experiments, it was found that surface-exposed histidines are modified in a concentration-dependent fashion and that this modification results in inactivation of the enzyme (k(inact) = 0.20 +/- 0.04 min(-)(1) mM(-)(1)). The DEPC modification was pH dependent and reversible by hydroxylamine, indicating that histidines are the sole residue being modified. As previously observed for heparinases I and II, substrate protection experiments slowed the inactivation kinetics, suggesting that the modified residue(s) was (were) in or proximal to the active site of the enzyme. Proteolytic mapping experiments, taken together with site-directed mutagenesis studies, confirm the chemical modification experiments and point to two histidines, histidine 295 and histidine 510, as being essential for heparinase III enzymatic activity.

Tumor cell surface heparan sulfate as cryptic promoters or inhibitors of tumor growth and metastasis

“…Recombinant Hep I and III were expressed and purified to homogeneity, as described (9,10). The enzymes were incubated with endotoxin removal resin (Associates of Cape Cod) to ensure its removal.…”

Amyloid Formation in Human Islets Is Enhanced by Heparin and Inhibited by Heparinase

“…To determine whether removal of cell surface heparan sulfate proteoglycans by heparinase treatment could prevent islet amyloid formation, human islets were cultured in 22 mM glucose in the absence or presence of heparinase III, which preferentially digests poorly sulfated cell-surface HSPGs (38). A high glucose concentration (22 mM) in culture was used to enhance amyloid formation (39) to facilitate detection of any protective action of heparinase treatment.…”

Biochemical Characterization of the Chondroitinase B Active Site

“…Because the H272A mutation shows a 6.5-fold decrease in k cat , this residue can potentially be involved in proton abstraction (Table I). Histidine has been implicated in the enzymatic degradation of other glycosaminoglycan-degrading enzymes, including Group B streptococcal hyaluronate lyase and heparinases I, II, and III (16,31,32). However, because the enzyme activity is not completely ablated, another residue may also be involved in abstraction of the C-5 proton.…”

“…PCR Site-directed Mutagenesis of Chondroitinase B-Lys-250, Arg-271, His-272, Glu-333, Arg-363, and Arg-364 were mutated to alanine using overlap extension PCR for 15 cycles (16). The primer sequences for each of the mutants are listed below.…”