Kinetic Characterization of Spinach Leaf Sucrose-Phosphate Synthase

Abstract

The spinach (Spinacia oleracea) leaf sucrose-phosphate synthase was partially purified via DEAE-cellulose chromatography, and its kinetic properties were studied. Fructose-6-phosphate saturation curves were sigmoidal, while UDPglucose saturation curves were hyperbolic. At subsaturating concentrations of fructose-6-phosphate, 1,5 anhydroglucitol-6-phosphate had a stimulatory effect on enzyme activity, suggesting multiple and interacting fructose-6-phosphate sites on sucrose-phosphate synthase. The concentrations required for 50% of maximal activity were 3.0 millimolar and 1.3 millimolar, respectively, for fructose-6-phosphate and UDPglucose. The enzyme was not stimulated by divalent cations. Inorganic phosphate proved to be a potent inhibitor, particularly at low concentrations of substrate. Phosphate inhibition was competitive with UDPglucose, and its K_i was determined to be 1.75 millimolar. Sucrose phosphate, the product of the reaction, was also shown to be a competitive inhibitor towards UDPglucose concentration and had K_i of 0.4 millimolar. The kinetic results suggest that spinach leaf sucrose-phospahte synthase is a regulatory enzyme and that its activity is modulated by the concentrations of phosphate, fructose-6-phosphate, and UDPglucose occurring in the cytoplasm of the leaf cell.