Hannesson H H, Hagner-McWhirter A, Tiedemann K, Lindahl U, Malmström A
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Biochem J. 1996 Jan 15;313 ( Pt 2)(Pt 2):589-96. doi: 10.1042/bj3130589.
The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ([GlcA(beta 1-->3)GalNAc(beta 1-->4)]n) backbone, to which 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 sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units. A Km of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80 x 10(-6) M) obtained for dermatan (desulphated dermatan sulphate). Vmax was about 4 times higher with dermatan than with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-3H]glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme. Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O 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 3H label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed.
大肠杆菌K4的荚膜多糖由一个软骨素（[GlcA(β1→3)GalNAc(β1→4)]n）主链组成，β-D-呋喃果糖单元连接在D-葡萄糖醛酸（GlcA）残基的C-3位上。通过温和酸水解去除果糖单元，得到了一种用于GlcA C-5差向异构酶的底物，该酶参与硫酸皮肤素生物合成过程中L-艾杜糖醛酸（IdoA）单元的生成。将该底物与溶解的成纤维细胞微粒体酶在3H2O存在下孵育，导致六糖醛酸单元的C-5位掺入了氚。测定得到的Km值为67×10-6 M六糖醛酸（相当于二糖单元），这与硫酸皮肤素（去硫酸化硫酸皮肤素）得到的Km值（80×10-6 M）相似。硫酸皮肤素的Vmax约为K4底物的4倍。用D-[5-3H]葡萄糖孵育细菌后分离得到的去果糖基K4多糖，与差向异构酶反应时会释放出3H2O，因此可用于该酶的测定。在3H2O存在下，将K4底物与溶解的微粒体差向异构酶孵育6小时，导致约5%的IdoA形成，并且GlcA和IdoA中的3H含量大致相等。对硫酸皮肤素进行相应孵育，得到约22%的GlcA，其中几乎包含了所有的3H标记。这些结果初步用双碱基反应机制来解释，该机制涉及一个单质子L-艾杜糖特异性碱基和一个多质子D-葡萄糖醛酸特异性碱基。尽管观察到有一些由两个或三个连续含IdoA的二糖单元形成，但该酶产生的大多数IdoA残基是单个出现的。
