Histidine 295 and histidine 510 are crucial for the enzymatic degradation of heparan sulfate by heparinase III.
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.
Pojasek
K
K
Division of Bioengineering & Environmental Health, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Shriver
Z
Z
Hu
Y
Y
Sasisekharan
R
R
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005417:Flavobacterium; D006497:Heparan Sulfate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D016297:Mutagenesis, Site-Directed; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D006497:Heparan Sulfate; D006639:Histidine; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase