The role of sulfhydryl groups in the regulation of spinach leaf sucrose phosphate synthase

Abstract

The activation of spinach leaf sucrose phosphate synthase by glucose 6-phosphate (Glc6 P) was labile in the absence of dithiothreitol, whereas enzyme activity in the absence of activator remained stable. The loss of regulation by Glc6 P proceeded more rapidly by treatment with 24 mM H 2O 2 and was reversible by 2.5 mM dithiothreitol. Titration of the enzyme with the sulfhydryl reagents N-ethylmaleimide or p-chloromercuribenzenesulfonic acid (0–50 μM) also resulted in elimination of Glc6 P activation and most of the inorganic phosphate inhibition without eliminating catalytic activity. The results suggested that Glc6 P and phosphate bind to a modifier site that is distinct from the catalytic site. The modifier site apparently contains essential and accessible sulfhydryl groups, while the catalytic site does not. Even though sulfhydryl reagents did not eliminate catalysis, changes in substrate affinities occurred. Treatment with 50 μM p-chloromercuribenzenesulfonic acid lowered the K m (fructose 6-phosphate) from 3.0 to 1.0 mM and increased the K m (uridine diphosphate glucose) from 3.0 to 8.9 mM without affecting the V max. Spinach leaf sucrose phosphate synthase extracted in the absence of reductant from leaves in the light (at 15:00 h) or in the dark (at 03:00 h) exhibited similar Glc6 P activation, suggesting that the light/dark status of leaves does not affect the redox status of essential sulfhydryl groups of the modifier site.