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 synthasefrom rice grains by free metal ions was studied. Decreasingsucrose synthase activities in the order of metal ions (Cu²⁺>> Zn²⁺ Ni²⁺ > Fe²⁺; 15·4% residual activitywith 30 µM Cu²⁺) as well as inhibition by diethyl pyrocarbonate(27% residual activity at pH 7·2 and 43 µM diethylpyrocarbonate) provided evidence that histidyl residues areimportant for sucrose synthase activity. Chelated metal ions,due to the geometric restriction of the reagent, gave a lesspronounced inhibitory effect (11·7% residual activitywith 100 µM Cu²⁺), but suggested that surface-accessiblehistidine residues are probably involved. Inhibition of sucrosesynthase could always be prevented by metal ion scavengers ethylenediaminetetra-aceticacid (EDTA), dithiothreitol (DTT), mercaptoethanol, reducedglutathione, imidazole and histidine]. Sucrose synthase inhibitedby free and chelated Cu²⁺, respectively, could be partly (60%)reactivated by EDTA. These results led to a topographical analysisof histidines on the surface of the homotetrameric protein byimmobilized metal ion chromatography (IMAC). From the orderby which sucrose synthase was bound to immobilized chelatedmetal ions in the presence of 1 mM imidazole (Cu²⁺ > Ni²⁺> Zn²⁺ = Co²⁺), it could be concluded that the enzyme hasat least 5–7 surface-accessible histidines. Sucrose synthasecould not be eluted from a Cu²⁺ column by an increasing imidazolegradient. These results are of particular interest for the furtherpurification of sucrose synthase(s), as well as for the evaluationof cloning and expression strategies using poly-hLstidine tails. glycosyltransferase IMAC nucleotide sugars sucrose synthase