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 synthase activities in the order of metal ions (Cu2+ >> Zn2+ > or = Ni2+ > Fe2+; 15.4% residual activity with 30 microM Cu2+) as well as inhibition by diethyl pyrocarbonate (27% residual activity at pH 7.2 and 43 microM diethyl pyrocarbonate) provided evidence that histidyl residues are important for sucrose synthase activity. Chelated metal ions, due to the geometric restriction of the reagent, gave a less pronounced inhibitory effect (11.7% residual activity with 100 microM Cu2+), but suggested that surface-accessible histidine residues are probably involved. Inhibition of sucrose synthase could always be prevented by metal ion scavengers [ethyl-enediaminetetra-acetic acid (EDTA), dithiothreitol (DTT), mercaptoethanol, reduced glutathione, imidazole and histidine]. Sucrose synthase inhibited by free and chelated Cu2+, respectively, could be partly (60%) reactivated by EDTA. These results led to a topographical analysis of histidines on the surface of the homotetrameric 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+ = Co2+), it could be concluded that the enzyme has at least 5-7 surface-accessible histidines. Sucrose synthase could not be eluted from a Cu2+ column by an increasing imidazole gradient. These results are of particular interest for the further purification of sucrose synthase(s), as well as for the evaluation of cloning and expression strategies using polyhistidine tails.
Elling
L
L
Institute for Enzyme Technology, Heinrich Heine University Düsseldorf in the Research Centre Jülich, FRG.
D002413:Cations, Divalent / Q000494:pharmacology*; D002846:Chromatography, Affinity; D003300:Copper / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000302:isolation & purification; D005978:Glutathione / Q000494:pharmacology; D006639:Histidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D012275:Oryza / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology
D002413:Cations, Divalent; D007093:Imidazoles; D006639:Histidine; D008623:Mercaptoethanol; D003300:Copper; C029899:imidazole; D004492:Edetic Acid; D005964:Glucosyltransferases; C022699:sucrose synthase; D005978:Glutathione; D003545:Cysteine; D004047:Diethyl Pyrocarbonate; D004229:Dithiothreitol