title: Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates.
abstract: The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-(3)H]glucose. Following complete or partial ( approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.SO(3)complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme. Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its K(m)value, which was significantly lower than that of its partially N-acetylated counterpart. The V(max)values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [(3)H]heparin used in our previous studies. Surprisingly, the apparent K(m)values for all three substrates increased with increasing enzyme concentration. The reason for this phenomenon is not entirely clear at present. Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent K(m)values with enzyme concentration precluded a detailed comparison of their kinetic properties.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 582, S-751 23, Uppsala, Sweden,
authors.forename: A
authors.initials: A
authors.lastname: Hagner-McWhirter
authors.forename: H H
authors.initials: HH
authors.lastname: Hannesson
authors.forename: P
authors.initials: P
authors.lastname: Campbell
authors.forename: J
authors.initials: J
authors.lastname: Westley
authors.forename: L
authors.initials: L
authors.lastname: Rodén
authors.forename: U
authors.initials: U
authors.lastname: Lindahl
authors.forename: J P
authors.initials: JP
authors.lastname: Li
mesh_terms: D000818:Animals; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002417:Cattle; D004926:Escherichia coli / Q000276:immunology / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006493:Heparin / Q000096:biosynthesis*; D006497:Heparan Sulfate / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D012515:Mast-Cell Sarcoma / Q000201:enzymology; D051379:Mice; D019042:Muscle Neoplasms / Q000201:enzymology; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D013464:Sulfuric Acids / Q000378:metabolism; D014316:Tritium
chemical_list: D002238:Carbohydrate Epimerases; D002250:Carbon Radioisotopes; D005947:Glucose; D006493:Heparin; D006497:Heparan Sulfate; D011134:Polysaccharides; D013464:Sulfuric Acids; D014316:Tritium; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase