title: Biosynthesis of heparin/heparan sulphate: mechanism of epimerization of glucuronyl C-5.
abstract: In the biosynthesis of heparin and heparan sulphate, D-glucuronic acid residues are converted into L-iduronic acid (IdoA) units by C-5 epimerization, at the polymer level. The reaction catalysed by the epimerase occurs by reversible abstraction and readdition of a proton at C-5 of target hexuronic acid residues, through a carbanion intermediate, with or without an inversion of configuration at C-5 [Prihar, Campbell, Feingold, Jacobsson, Jensen, Lindahl and Rodén (1980) Biochemistry 19, 495-500]. Incubation of chemically N-sulphated capsular polysaccharide from Escherichia coli K5 ([4GlcAbeta1-4GlcNSO(3)alpha1-](n)), or of O-desulphated heparin (predominantly [4IdoAalpha1-4GlcNSO(3)alpha1-](n)) with purified C-5 epimerase from bovine liver, resulted in the interconversion of glucuronic acid and IdoA residues, which reached equilibrium (30-40% IdoA/total hexuronic acid) after approx. 1 h of incubation. Similar incubations performed in the presence of (3)H(2)O resulted in progressive labelling at C-5 of the target hexuronic acid units of either substrate polysaccharide. Contrary to chemical D-gluco/L-ido equilibrium, established within 1 h of incubation, the accumulation of (3)H label continued for at least 6 h. This isotope effect suggests that the second stage of the reaction, i.e. the re-addition of a proton to the carbanion intermediate, is the rate-limiting step of the overall process. Analysis of the 5-(3)H-labelled polysaccharide products showed that the (3)H was approximately equally distributed between glucuronic acid and IdoA units, irrespective of incubation time (from 15 min to 72 h) and of the relative proportions of the two epimers in the substrate. This finding points to a catalytic mechanism in which the abstraction and re-addition of C-5 protons are effected by two polyprotic bases, presumably lysine residues. Previous experiments relating to the biosynthesis of dermatan sulphate were similarly interpreted in terms of a two-base epimerization mechanism but differed from the present findings by implicating one monoprotic and one polyprotic base function [Hannesson, Hagner-McWhirter, Tiedemann, Lindahl and Malmström (1996) Biochem. J. 313, 589-596].
authors.affiliation: Department of Medical Biochemistry, Section for Medical Biochemistry, University of Uppsala, Biomedical Center, SE-751 23 Uppsala, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Hagner-Mcwhirter
authors.forename: U
authors.initials: U
authors.lastname: Lindahl
authors.forename: J p
authors.initials: Jp
authors.lastname: Li
mesh_terms: D000818:Animals; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002417:Cattle; D004926:Escherichia coli; D020723:Glucuronic Acid / Q000378:metabolism*; D006493:Heparin / Q000096:biosynthesis* / Q000737:chemistry; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000737:chemistry; D007067:Iduronic Acid / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002238:Carbohydrate Epimerases; D020723:Glucuronic Acid; D006493:Heparin; D006497:Heparan Sulfate; D007067:Iduronic Acid; D011135:Polysaccharides, Bacterial; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase