The mechanism of dextransucrase action: Direction of dextran biosynthesis

Abstract

The mechanism for the biosynthesis of dextran by dextransucrase from Leuconostoc mesenteroides NRRL-B512F has been studied. Dextransucrase attached to cells and dextransucrase insolubilized on Bio-Gel beads have been obtained. It was found that these forms become labeled when incubated with a pulse of [ 14C]sucrose. The label can be released from cells and from Bio-Gel by adjusting the pH to 2 and heating to 95 °C for 10 min. Two types of labeled carbohydrates were found to be released, glucose and dextran. The direction of the biosynthesis was determined by pulse and chase experiments with [ 14C]sucrose. The labeled dextran was isolated, reduced, and acid hydrolyzed. The hydrolytic products, glucose and sorbitol, were separated by paper chromatography, and their radioactivities were determined. The ratio of the labeled sorbitol to labeled glucose for the pulse of Bio-Gel-dextransucrase was 1:1 and for the chase 1:100. It was concluded that dextran is biosynthesized by the transfer of glucose from sucrose to the reducing end of the growing dextran chain. This was confirmed by the hydrolysis of pulsed dextran by glycoamylase. The total radioactivity in the residual dextran did not change during the hydrolysis and labeled glucose could not be detected. It is proposed that dextransucrase forms an enzymatically active covalent complex with glucose and dextran and that the glucose is inserted between the enzyme and the dextran by a nucleophilic attack of the C 6—OH of glucose onto C 1 of the dextran forming an α-1 → 6 glucosidic bond. This releases one of the nucleophilic groups at the active site which attacks sucrose to give an enzyme-glucosyl complex. The C 6—OH of this glucose then repeats the process of attacking C 1 of dextran. Dextran is built up by extrusion from the enzyme when glucose units are transferred from sucrose to the active site and inserted between the enzyme and the reducing end of the dextran polymer.