Enzymic mechanism of starch synthesis in ripening rice grains: VII. Purification and enzymic properties of sucrose synthetase

Abstract

Sucrose synthetase (UDP-glucose: d-fructose-2-glucosyltransferase, EC 2.4.1.13) from ripening rice seeds was purified by ammonium sulfate fractionation and column chromatography of microgranular DEAE-cellulose (DE-32) and Neusilin (MgO· Al 2O 3·2SiO 2). An enzyme preparation obtained was homogeneous as examined by polyacrylamide gel electrophoresis. The enzyme, having a molecular weight, 4.0 × 10 5, consists of 4 identical subunits, each having a molecular weight, 1.0 × 10 5. Examination of reaction kinetics of both sucrose synthesis and cleavage catalyzed by sucrose synthetase revealed that the rate of synthesis follows a Michaelis-Menten equation having the following parameters: K m (fructose) UDP-glucose, 6.9 m m; K m (fructose) ADP-glucose, 40 m m; K m (UDP-glucose), 5.3 m m; and K m (ADP-glucose), 3.8 m m. The cleavage reaction yielded the following values: K m (UDP), 0.8 m m; K m (ADP), 3.3 m m; and K m (sucrose) UDP, 290 m m. In the latter reaction the rate deviated from the Michaelis equation when ADP was used as the glucose acceptor, the n value being 1.6 by the Hill plot analysis and S 0.5(sucrose) ADP, 400 m m. At high concentration of ADP the cleavage reaction was inhibited, while the synthesis reaction was inhibited with high concentrations of fructose.