Heparinase I from Flavobacterium heparinum: the role of the cysteine residue in catalysis as probed by chemical modification and site-directed mutagenesis.
Heparinase I (heparin lyase I, EC 4.2.2.7), a heparin-degrading enzyme produced by Flavobacterium heparinum, is used to deheparinize blood following extracorporeal procedures in surgery and in other applications. The present study of mapping and characterization of the cysteines of heparinase I represents the first structural characterization of a heparinase. [3H]Iodoacetic acid labeling demonstrated that heparinase I has two free cysteines. One of the two cysteines is surface accessible and lies in a hydrophilic environment while the other is in a hydrophobic environment. Chemical modification of the cysteines, both in the presence and in the absence of heparin, suggests that the surface-accessible cysteine lies in or near the active site of heparinase I. Preferential reactivity of this cysteine with negatively charged sulfhydryl-modifying reagents and the cysteines' high reactivity to iodoacetic acid at pH 6.5 indicate that the surface-accessible cysteine is in a positively charged region. The surface-accessible cysteine (cysteine-135) was mapped as the active-site cysteine by radiolabeling with [3H]iodoacetic acid and by tryptic digestion and peptide sequencing. Site-directed mutagenesis of cysteine-135 to a serine or an alanine in r-heparinase I demonstrates that this cysteine is essential for enzymatic activity. However, replacement of the surface-inaccessible cysteine by a serine or alanine has no effect.
Sasisekharan
R
R
Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Leckband
D
D
Godavarti
R
R
Venkataraman
G
G
Cooney
C L
CL
Langer
R
R
D001483:Base Sequence; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005417:Flavobacterium / Q000201:enzymology*; D019763:Heparin Lyase; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D015335:Molecular Probes; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase; D003545:Cysteine