BIOSYNTHESIS OF HEPARIN - USE OF ESCHERICHIA-COLI K5 CAPSULAR POLYSACCHARIDE AS A MODEL SUBSTRATE IN ENZYMATIC POLYMER-MODIFICATION REACTIONS

A capsular polysaccharide from Escherichia coli K5 was previously found to have the same structure, [-(4)beta-GlcA(1)-->(4)alpha-GlcNAc(1)-](n), as that of the non-sulphated precursor polysaccharide in heparin biosynthesis [Vann, Schmidt, Jann & Jann (1981) Eur. J. Biochem. 116, 359-364]. The K5 polysaccharide was N-deacetylated (by hydrazinolysis) and N-sulphated, and was then incubated with detergent-solubilized enzymes from a heparin-producing mouse mastocytoma, in the presence of adenosine 3'-phosphate 5'-phospho[S-35]sulphate ([S-35]PAPS). Structural analysis of the resulting S-35-labelled polysaccharide revealed the formation of all the major disaccharide units found in heparin. The identification of 2-O-[S-35]sulphated IdoA (L-iduronic acid) as well as 6-O-[S-35]sulphated GlcNSO3 units demonstrated that the modified K5 polysaccharide served as a substrate in the hexuronosyl C-5-epimerase and the major O-sulphotransferase reactions involved in the biosynthesis of heparin. The GlcA units of the native (N-acetylated) E. coli polysaccharide were attacked by the epimerase only when PAPS was present in the incubations, whereas those of the chemically N-sulphated polysaccharide were epimerized also in the absence of PAPS, in accord with the nation that N-sulphate groups are required for epimerization. With increasing concentrations of PAPS, the mono-O-sulphated disaccharide unit -IdoA(2-OSO3)-GlcNSO3- was progressively converted into the di-O-sulphated species -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-. A small proportion of the S-35-labelled polysaccharide was found to bind with high affinity to the proteinase inhibitor antithrombin. This proportion increased with increasing concentration of PAPS up to a level corresponding to approximately 1-2% of the total incorporated S-35. The solubilized enzymes thus catalysed all the reactions required for the generation of functional antithrombin-binding sites.