Heparinase II from Flavobacterium heparinum. Role of histidine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.

The Journal of Biological Chemistry
Z ShriverR Sasisekharan

Abstract

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...Continue Reading

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Citations

Mar 7, 2012·Applied Microbiology and Biotechnology·C K M TripathiVikas Mishra
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