De novo biosynthesis of starch chains without a primer and the mechanism for its biosynthesis by potato starch-synthase Original Research Article

The starch-synthase enzymes used in this study were the second acetone precipitate and Fractions 21 and 23, , [Mukerjea, Ru.; Falconer, D. J.; Yoon, S.-H.; Robyt, J. F. Carbohydr. Res. 2010, 345, 1555–1563]. Fractions 21 and 23 had high specific activities of 544 and 944 International Units/mg, respectively. When the enzymes and buffer and substrate were treated with immobilized α-amylase and glucoamylase for 30 min, they all had the same activity, before and after treatment, indicating that the enzymes were free of putative primers and synthesized amylose chains de novo, without the addition of primers. Starch-synthase was immobilized and reacted with ADP-[14C]Glc; the immobilized enzyme was removed, washed and treated at pH 2 and 50 °C for 30 min, giving the release of 14C-d-glucopyranose and 14C-amylose, showing that during catalysis they were covalently attached to the enzyme active-site. Pulse and chase reactions of starch-synthase with ADP-[14C]Glc and ADPGlc, respectively, followed by reduction and acid hydrolysis of the starch-chain product, gave 14C-d-glucitol from the pulse reaction and a significant decrease of 14C-d-glucitol from the chase reaction, showing that the addition of d-glucose from ADPGlc was to the reducing-ends of the growing amylose chains. Reactions of four different concentrations of starch-synthase, with constant ADPGlc concentration and temperature, gave four amylose chains, each with different number average molecular weights that were inversely proportional to the concentration of the enzyme, indicating that the synthesis was processive. From the results, a two catalytic-site, insertion mechanism is proposed for the biosynthesis of starch chains.