Biosynthesis of Uridine Diphosphate d-Xylose: IV. MECHANISM OF ACTION OF URIDINE DIPHOSPHOGLUCURONATE CARBOXY-LYASE

Abstract

Abstract UDP-d-glucuronic acid labeled with tritium at either C-3, C-4, or C-5 was converted to UDP-d-xylose by UDP-glucuronate carboxy-lyase (EC 4.1.1.35) from wheat germ and Cryptococcus laurentii. A kinetic isotope effect (Vt/Vh) of 0.32 and 0.42 for the C. laurentii and wheat germ enzymes, respectively, was observed with the C-4-labeled substrate but not with the C-3- or C-5-labeled substrate. Labeled UDP-d-xylose obtained from UDP-d-glucuronic acid-5T was converted with phosphodiesterase to labeled α-d-xylosyl phosphate. The latter was oxidized with periodic acid to yield d-phosphodiglycolic aldehyde from C-1 and C-2 and from C-4 and C-5; hypobromite oxidation of the dialdehyde yielded d-phosphodiglycolic acid. Acid hydrolysis of this compound released unlabeled glyoxalic acid (which originated from C-1 and C-2) and labeled glycolic acid (from C-4 and C-5). The latter was converted to unlabeled glyoxalic acid upon oxidation with glycolic oxidase from spinach, which is specific for that hydrogen atom in glycolic acid sterically equivalent to the α-hydrogen of l-lactic acid. These results show that the configuration at C-5 changed from S to R during the decarboxylation of UDP-d-glucuronic acid. Since UDP-glucuronate carboxy-lyase requires NAD for activity, these data suggest that the reaction involves oxidation at C-4 to yield UDP-4-keto-d-glucuronic acid, followed by decarboxylation and formation of UDP-4-keto-d-xylose, which is then stereospecifically protonated at C-5 and reduced at C-4 to yield product. The presence of an isotope effect with UDP-d-glucuronic acid-4T means that either the initial oxidation or the final reduction is the rate-limiting step of the reaction; it is not possible to differentiate between these two possibilities on the basis of the available data.