Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Sulfur regulation of heparinase synthesis and sulfatase synthesis was studied in Flavobacterium heparinum. Heparinase synthesis was strongly repressed by sulfate and L-cysteine, while the activity of this enzyme showed little or no inhibition by these compounds. Heparinase was synthesized in the absence of heparin when L-methionine was used as the sole sulfur source. The sulfatases produced by F. heparinum, which include the sulfatases involved in heparin catabolism, were also studied. At least some of the sulfatase activity was regulated by sulfur compounds in a manner similar to heparinase regulation. L-Cysteic acid and taurine were not suitable sulfur sources to support the growth of F. heparinum.
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PMID:Sulfur regulation of heparinase and sulfatases in Flavobacterium heparinum. 396 13

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.
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PMID:Purification and characterization of heparinase from Flavobacterium heparinum. 396 88

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

In this study we have identified the primary heparin binding site of heparinase I (EC 4.2.2.7). Chemical and proteolytic digests of heparinase I were used in direct binding and competition assays, to map the regions of heparinase I that interact specifically with heparin. We find the heparin binding site contains two Cardin-Weintraub heparin binding consensus sequences and a calcium co-ordination consensus motif. We show that heparin binding to heparinase I is independent of calcium (Kd of 60 nm) and that calcium is able to activate heparinase I catalytically. We find that sulfhydryl selective labeling of cysteine 135 of heparinase I protects the lysines of the heparin binding sequence from proteolytic cleavage, suggesting the close proximity of the heparin binding site to the active site. Site-directed mutagenesis of H203A (contained in the heparin binding site) inactivated heparinase I; however, a H203D mutant retained marginal activity, indicating a role for this residue in catalysis. The above results taken together suggest that histidine 203 (hence the heparin binding site) is immediately adjacent to the scissile bond. We propose that the heparin binding site and active site are in close proximity to each other and that the calcium coordination motif, contained in the heparin binding site, may bridge heparin to heparinase I through calcium in a ternary complex during catalysis.
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PMID:Heparinase I from Flavobacterium heparinum. Mapping and characterization of the heparin binding domain. 862 11

We recently identified cysteine-135 as an important amino acid for heparinase I (EC 4.2.2.7) activity. In this study, we have identified a second residue critical for enzymatic activity. We observe concentration-dependent inactivation of heparinase I in the presence of reversible histidine-modifying diethyl pyrocarbonate (DEPC); 0.3 mM DEPC results in 95% of heparinase I inactivation in less than 3 min, and as low as 10 microM DEPC results in a 85% loss of heparinase I activity in 15 min. Heparinase I activity is restored following hydroxylamine treatment. This, along with other experiments, strongly suggests that the inactivation of heparinase I by DEPC is specific for histidine residues. Chemical modification, under nondenaturing conditions, of the histidines using nonradiolabeled and [14C]DEPC indicates that between one and two histidine residues are modified. Chemical modification of the surface-accessible histidines, in the presence and absence of heparin, suggests that the histidine(s) lie(s) in or near the active site of heparinase I. The wild-type heparinase I has four histidine residues; site-directed mutagenesis of H129A, H165A, and H339A did not affect enzyme activity and the kinetic parameters, suggesting that these residues are not essential for heparinase I activity. However, H203A inactivates heparinase I while a H203D mutant has residual activity, indicating a role of this residue in catalysis. We propose that histidine-203, contained in the heparin binding site, is immediately adjacent to cysteine-135, and these residues together form the catalytic domain of heparinase I.
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PMID:Heparinase I from Flavobacterium heparinum. Identification of a critical histidine residue essential for catalysis as probed by chemical modification and site-directed mutagenesis. 863 36

Using a macrophage cell line that constitutively expresses a human apolipoprotein E (apoE) cDNA, we have investigated the post-translational metabolism of endogenously produced apoE. Inhibition of lysosomal or cysteine proteases led to significant inhibition of apoE degradation but did not increase apoE secretion, indicating that cellular degradation is not limiting for apoE secretion in macrophages. Treatment of macrophages with inhibitors of proteoglycan synthesis (4-methylumbelliferyl-beta-D-xyloside) or sulfation (sodium chlorate) enhanced the release of apoE from cells and significantly attenuated the increase in secretion produced by incubation with phosphatidylcholine vesicles (PV). These observations suggested that a significant fraction of the apoE retained by cells (and released by incubation with PV) was associated with proteoglycans. Treatment of cells with exogenous heparinase led to a greater than 4-fold increase in apoE secretion and similarly attenuated the response to PV, suggesting that apoE was trapped in an extracellular proteoglycan matrix. This conclusion was confirmed in studies showing that PV could enhance the release of apoE from cells during an incubation at 4 degrees C, but this enhanced release was abolished in proteoglycan-depleted cells. Incubation with lactoferrin at 4 or 37 degrees C produced a similar decrement in cellular apoE, again indicating the existence of a cell surface pool of apoE. Pulse-chase studies showed that the apoE trapped in the proteoglycan matrix was susceptible to rapid cellular degradation such that net synthesis of apoE (secreted plus cell-associated) was increased significantly in proteoglycan-depleted cells compared with control cells as early as 45 min during a chase period.
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PMID:Cell surface proteoglycans modulate net synthesis and secretion of macrophage apolipoprotein E. 866 12

Heparinases are bacterial enzymes that are powerful tools to study the physiological roles of heparin-like complex polysaccharides. In addition, heparinases have significant therapeutic applications. We had proposed earlier that cysteine 135 and histidine 203 together form the catalytic domain in heparinase I. We had also identified a heparin binding domain in heparinase I containing two positively charged clusters HB-1 and HB-2 in a primary heparin binding site and other positively charged residues in the vicinity of cysteine 135. In this study, through systematic site-directed mutagenesis studies, we show that the alteration of the positive charge of the HB-1 region has a pronounced effect on heparinase I activity. More specifically, site-directed mutagenesis of K199A (contained in HB-1) results in a 15-fold reduction in catalytic activity, whereas a K198A mutation (also in HB-1) results in only a 2- to 3-fold reduction in heparinase I activity. A K132A mutation, in close proximity to cysteine 135, also resulted in reduced (8-fold) activity. Heparin affinity chromatography experiments indicated moderately lowered binding affinities for the K132A, K198A, and the K199A mutant enzymes. The above results, taken together with our previous observations, lead us to propose that the positively charged heparin binding domain provides the necessary microenvironment for the catalytic domain of heparinase I. The dominant effect of lysine 199 suggests an additional, more direct, role in catalysis for this residue.
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PMID:Heparinase I from Flavobacterium heparinum. Role of positive charge in enzymatic activity. 941 72

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.
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PMID:Heparinase II from Flavobacterium heparinum. Role of cysteine in enzymatic activity as probed by chemical modification and site- directed mutagenesis. 972 10

A novel type of heparinase (heparin lyase, no EC number) has been purified from Bacteroides stercoris HJ-15, isolated from human intestine, which produces three kinds of heparinases. The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, DEAE-cellulose, CM-Sephadex C-50, hydroxyapatite, and HiTrap SP chromatographies with a final specific activity of 19.5 mmol/min/mg. It showed optimal activity at pH 7.2 and 45 degrees C and the presence of 300 mM KCl greatly enhanced its activity. The purified enzyme activity was inhibited by Cu(2+), Pb(2+), and some agents that modify histidine and cysteine residues, and activated by reducing agents such as dithiothreitol and 2-mercaptoethanol. This purified Bacteroides heparinase is an eliminase that shows its greatest activity on bovine intestinal heparan sulfate, and to a lesser extent on porcine intestinal heparan sulfate and heparin. This enzyme does not act on acharan sulfate but de-O-sulfated acharan sulfate and N-sulfoacharan sulfate were found to be poor substrates. The substrate specificity of this enzyme is similar to that of Flavobacterial heparinase II. However, an internal amino acid sequence of the purified Bacteroides heparinase shows significant (73%) homology to Flavobacterial heparinase III and only 43% homology to Flavobacterial heparinase II. These findings suggest that the Bacteroidal heparinase is a novel enzyme degrading GAGs.
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PMID:Purification and characterization of a novel heparinase from Bacteroides stercoris HJ-15. 1092 Feb 69

Two novel acharan sulfate lyases (ASL1 and ASL2: no EC number) have been purified from Bacteroides stercoris HJ-15 which was isolated from human intestinal bacteria with glycosaminoglycan (GAG) degrading enzymes. These enzymes were purified to apparent homogeneity by a combination of QAE-cellulose, DEAE-cellulose, carboxymethyl-Sephadex C-50, hydroxyapatite and HiTrap SP Sephadex C-25 column chromatography with the final specific activity of 50.5 and 76.7 micromol.min-1.mg-1, respectively. Both acharan sulfate lyases are single subunits of 83 kDa by SDS/PAGE and gel filtration. ASL1 showed optimal activity at pH 7.2 and 45 degrees C. ASL1 activity was inhibited by Cu2+, Ni2+ and Co2+, but ASL2 activity was inhibited by Cu2+, Ni2+and Pb2. Both enzymes were slightly inhibited by some agents that modify histidine and cysteine residues, but activated by reducing agents such as DL-dithiothreitol and 2-mercaptoethanol. Both purified bacteroidal acharan sulfate lyases acted to the greatest extent on acharan sulfate, and to a lesser extents on heparan sulfate and heparin. They did not act on de-O-sulfated acharan sulfate. These findings suggest that the biochemical properties of these purified acharan sulfate lyases are different from those of the previously purified heparin lyases, but these enzymes belong to heparinase II.
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PMID:Purification and characterization of acharan sulfate lyases, two novel heparinases, from Bacteroides stercoris HJ-15. 1132 84


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