Further Studies on the Mechanism of Action of UDP‐Apiose/UDP‐Xylose Synthase from Cell Cultures of Parsley

Abstract

1. When uridine diphopho‐d‐[U‐14C]xylose was incubated with the UDP‐apiose/UDP‐xylose synthase from cell suspension cultures of parsley no radioactivity was detected in UDP‐apiose (UDP‐Api). 2. UDP‐d‐glucuronic acid (UDP‐GlcUA) labeled with tritum at either C‐4 or C‐5 was converted to UDP‐Api/UDP‐Xyl synthase. Free apiose was reduced to apiitol which was oxidized with periodic acid to glycolic acid. In both cases oxidation of glycolic acid with glycollate oxidase from spinach to glyoxalic acid released all the tritium into the water. This result proves that during the hydride shift from UDP‐[4‐3H]GlcUA tritium is transfered only to the pro‐R position at C‐3′ of apiose and that inversion of configuration occurs at C‐4 of apiose in the synthase reaction. 3. When the UDP‐Api/UDP‐Xyl synthase reaction was carried out in a 3H1HO/2H2O mixture, a total of 1.5 mol 3H per mol apiose was incorporated with correction made for the product isotope effect. By degradation of apiose it was proved that 1 mol 3H was located at C‐4 and 0.5 3H per mol sugar at C‐3′ of apiose. 4. After addition of NaB3H4 to the enzyme incubation with UDP‐d‐[U‐14C]GlcUA and hydrolysis of the nucleiotide sugars, arabinose, xylose and glucuronic acid with a constant 3H/14C ratio were isolated after extensive purification. it was shown by degradation of xylose that over 90% of the tritium was located at C‐4 of this sugar. These results prove the existence of a 4‐keto intermediate and the formation of UDP‐4‐keto‐glucuronic acid as an intermediate in the decarboxylation step. 5. The results presented can best be explained by an aldolase type mechanism for the UDP‐Api/UDP‐Xyl synthase reaction. Copyright © 1973, Wiley Blackwell. All rights reserved