EFFECT OF METAL-IONS ON SUCROSE SYNTHASE FROM RICE GRAINS - A STUDY ON ENZYME-INHIBITION AND ENZYME TOPOGRAPHY

The inhibition of the plant glycosyltransferase sucrose synthase from rice grains by free metal ions was studied. Decreasing sucrose synthas e activities in the order of metal ions (Cu2+ >> Zn2+ greater than or equal to Ni2+ > Fe2+; 15.4% residual activity with 30 mu M Cu2+) as we ll as inhibition by diethyl pyrocarbonate (27% residual activity at pH 7.2 and 43 mu M diethyl pyrocarbonate) provided evidence that histidy l residues are important for sucrose synthase activity, Chelated metal ions, due to the geometric restriction of the reagent, gave a less pr onounced inhibitory effect (11.7% residual activity with 100 mu M Cu2), but suggested that surface-accessible histidine residues are probab ly involved, Inhibition of sucrose synthase could always be prevented by metal ion scavengers [ethylenediaminetetra-acetic acid (EDTA), dith iothreitol (DTT), mercaptoethanol, reduced glutathione, imidazole and histidine]. Sucrose synthase inhibited by free and chelated Cu2+, resp ectively, could be partly (60%) reactivated by EDTA, These results led to a topographical analysis of histidines on the surface of the homot etrameric protein by immobilized metal ion chromatography (IMAC), From the order by which sucrose synthase was bound to immobilized chelated metal ions in the presence of 1 mM imidazole (Cu2+ > Ni2+ > Zn2+ = Co 2+), it could be concluded that the enzyme has at least 5-7 surface-ac cessible histidines. Sucrose synthase could not be eluted from a Cu2column by an increasing imidazole gradient, These results are of parti cular interest for the further purification of sucrose synthase(s), as well as for the evaluation of cloning and expression strategies using poly-histidine tails.