Heparinase II from Flavobacterium heparinum. Role of cysteine in enzymatic activity as probed by chemical modification and site- directed mutagenesis.
Heparinase II (no EC number) is one of three lyases isolated from Flavobacterium heparinum that degrade heparin-like complex polysaccharides. Heparinase II is unique among the heparinases in that it has broad substrate requirements and possesses the ability to degrade both heparin and heparan sulfate-like regions of glycosaminoglycans. This study set out to investigate the role of cysteines in heparinase II activity. Through a series of chemical modification experiments, it was found that one of the three cysteines in heparinase II is surface-accessible and possesses unusual chemical reactivity toward cysteine-specific chemical modifying reagents. Substrate protection experiments suggest that this surface-accessible cysteine is proximate to the active site, since addition of substrate shields the cysteine from modifying reagents. The cysteine, present in an ionic environment, was mapped by radiolabeling with N-[3H]ethylmaleimide and identified as cysteine 348. Site-directed mutagenesis of cysteine 348 to an alanine resulted in loss of activity toward heparin but not heparan sulfate, indicating that cysteine 348 is required for heparinase II activity toward heparin but is not essential for the breakdown of heparan sulfate. Furthermore, we show in this study that cysteine 164 and cysteine 189 are functionally unimportant for heparinase II.
Shriver
Z
Z
Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Hu
Y
Y
Pojasek
K
K
Sasisekharan
R
R
D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005417:Flavobacterium / Q000201:enzymology*; D006493:Heparin / Q000494:pharmacology; D019763:Heparin Lyase / Q000378:metabolism; D006497:Heparan Sulfate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments; D011133:Polysaccharide-Lyases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D003545:Cysteine; D006493:Heparin; D019763:Heparin Lyase; D006497:Heparan Sulfate; D008666:Metalloendopeptidases; D010446:Peptide Fragments; D011133:Polysaccharide-Lyases; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C039830:peptidyl-Lys metalloendopeptidase; C114663:heparinase II; C021349:heparitinsulfate lyase