Glycobiology, 2000, Vol. 10, No. 2 159-171
© 2000 Oxford University Press
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
Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 582, S-751 23, Uppsala, Sweden, 2Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA, and 3Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
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-[5-3H]glucose. Following complete or partial (~50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine·SO3 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 Km value, which was significantly lower than that of its partially N-acetylated counterpart. The Vmax values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [3H]heparin used in our previous studies. Surprisingly, the apparent Km 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 Km values with enzyme concentration precluded a detailed comparison of their kinetic properties.
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