Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.2.2.7 (heparinase)
 1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Heparinase I from Flavobacterium heparinum: the role of the cysteine residue in catalysis as probed by chemical modification and site-directed mutagenesis. 757 49

A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/microg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 degrees C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co2+, Mn2+ ions, and reducing agents (beta-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K (m) and V (max) as 2.2 x 10(-5 )M and 30.8 mM min(-1), respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.
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PMID:Purification and characterization of a novel heparin degrading enzyme from Aspergillus flavus (MTCC-8654). 1921 96

An intracellularly produced constitutive heparinase was isolated from the periplasmic space of Acinetobacter calcoaceticus by freeze fracturing and purified 51.2-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 41 IU/mug protein with a 120000Da molecular mass. The enzyme activity was maximum at 35 degrees C in the presence of 250mM NaCl at pH 7.5. The enzyme activity was inhibited in the presence of Ba(2+), Hg(2+), Cd(2+), IAA and DEPC, and enhanced by the presence of Cu(2+), Fe(2+) ions and reducing agents. Inhibition of enzyme activity by iodoacetic acid and enhancement of enzyme activity in the presence of reducing agents indicated that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The affinity of the enzyme for different glycosaminoglycans studied varied and showed high affinity for heparin with a K(m) value of 0.026mM. In situ gel digestion of the purified protein with trypsin did not show any homology with heparinase I. Depolymerization of heparin and fractionation of the oligosachharides yielded heparin disaccharides as main product. This suggests a catalytic similarity and structural dissimilarity of heparinase from Acinetobacter with heparinase I.
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PMID:Rapid purification and characterization of a novel heparin degrading enzyme from Acinetobacter calcoaceticus. 1942 46