Heparinase II from Flavobacterium heparinum. Role of histidine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.
The three heparinases derived from Flavobacterium heparinum are powerful tools for studying heparin-like glycosaminoglycans in major biological processes, including angiogenesis and development. Heparinase II is unique among the three enzymes because it is able to catalytically cleave both heparin and heparan sulfate-like regions of heparin-like glycosaminoglycans. Toward understanding the catalytic mechanism of heparin-like glycosaminoglycan degradation by heparinase II, we set out to investigate the role of the histidines of heparinase II in catalysis. We observe concentration-dependent inactivation of heparinase II in the presence of the reversible histidine-modifying reagent diethylpyrocarbonate (DEPC). With heparin as the substrate, the rate constant of inactivation was found to be 0.16 min-1 mM-1; with heparan sulfate as the substrate, the rate constant was determined to be 0.24 min-1 mM-1. Heparinase II activity is restored following hydroxylamine treatment. This, along with other experiments, strongly suggests that the inactivation of heparinase II by DEPC is specific for histidine residues and that three histidines are modified by DEPC. Substrate protection experiments show that heparinase II preincubation with heparin followed by the addition of DEPC resulted in a loss of enzymatic activity toward heparan sulfate but not heparin. However, heparinase II preincubation with heparan sulfate was unable to protect heparinase II from DEPC inactivation for either of the substrates. Proteolytic mapping studies with Lys-C were consistent with the chemical modification experiments and identified histidines 238, 451, and 579 as being important for heparinase II activity. Further mapping studies identified histidine 451 as being essential for heparin degradation. Site-directed mutagenesis experiments on the 13 histidines of heparinase II corroborated the chemical modification and the peptide mapping studies, establishing the importance of histidines 238, 451 and 579 in heparinase II activity.
Shriver
Z
Z
Whitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Hu
Y
Y
Sasisekharan
R
R
D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004789:Enzyme Activation; D005417:Flavobacterium / Q000201:enzymology*; D006493:Heparin / Q000378:metabolism; D019763:Heparin Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed
D004047:Diethyl Pyrocarbonate; D006493:Heparin; D019763:Heparin Lyase; D006497:Heparan Sulfate; D006639:Histidine; D015335:Molecular Probes