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  • Title: Biosynthesis of heparin. Substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase.
    Author: Jacobsson I, Lindahl U, Jensen JW, Rodén L, Prihar H, Feingold DS.
    Journal: J Biol Chem; 1984 Jan 25; 259(2):1056-63. PubMed ID: 6420398.
    Abstract:
    The substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase from a mouse mastocytoma was examined to determine the effects of N-acetyl and O-sulfate groups on substrate recognition by the enzyme. [5-3H]Glucuronosyl-labeled heparosan N-sulfate was prepared enzymatically and was modified chemically by partial N-desulfation and N-acetylation. After enzymatic release of tritium, the location of remaining label was determined by deaminative cleavage and analysis of resulting di-, tetra-, and higher oligosaccharides. This analysis indicated that a D-glucuronosyl residue is recognized as a substrate if it is linked at C-1 to an N-acetylated glucosamine residue and at C-4 to an N-sulfated unit. However, the reverse structure, in which the D-glucuronosyl moiety is bound at C-1 to an N-sulfated residue and at C-4 to N-acetylated glucosamine, is not a substrate. Similar studies with O-sulfated heparin intermediates showed that O-sulfate groups either at C-2 of the L-iduronosyl moieties or at C-6 of vicinal D-glucosaminyl moieties prevent 5-epimerization. These findings were confirmed by studies of the reverse reaction, in which tritium was incorporated from 3H2O into partially O-desulfated heparin and the location of incorporated radioactivity was determined. These and more direct experiments corroborated the previous conclusion that the L-iduronosyl moieties are formed after N-sulfation but before O-sulfation. Assessment of the influence of substrate size on the reaction further showed that a large substrate is preferred; an octasaccharide released tritium at a rate approximately 10% of that observed for the parent polysaccharide, and some release occurred also with smaller oligosaccharides.
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