BIOSYNTHESIS OF DERMATAN SULFATE - DEFRUCTOSYLATED ESCHERICHIA-COLI K4 CAPSULAR POLYSACCHARIDE AS A SUBSTRATE FOR THE D-GLUCURONYL C-5 EPIMERASE, AND AN INDICATION OF A 2-BASE REACTION-MECHANISM

The capsular polysaccharide from Escherichia coli K4 consists of a cho ndroitin {[GlcA(beta 1 --> 3)GalNAc(beta 1 --> 4)](n)} backbone, to wh ich beta-fructofuranose units are linked to C-3 of D-glucuronic acid ( GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphat e biosynthesis. Incubation of this substrate with solubilized fibrobla st microsomal enzyme in the presence of (H2O)-H-3 resulted in the inco rporation of tritium at C-5 of hexuronyl units. A K-m of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, whi ch is similar to that (80 x 10(-6) M) obtained for dermatan (desulphat ed dermatan sulphate). V-max was about 4 times higher with dermatan th an with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-H-3]glucose released (H2O)-H-3 on reaction with the epimerase, and thus could be used to assay the e nzyme. Incubation of a K4 substrate with solubilized microsomal epimer ase for 6 h in the presence of (H2O)-H-3 resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA. A corresponding incubation of dermatan yielded approx. 22% GlcA, which contained virtually all the H-3 label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monop rotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, althoug h some formation of two or three consecutive IdoA-containing disacchar ide units was observed.