MECHANISM OF REDUCTIVE ACTIVATION OF POTATO-TUBER ADP-GLUCOSE PYROPHOSPHORYLASE

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase activity is activated by a incubation with ADP-glucose and dithiothrei tol or by ATP, glucose-1-phosphate, Ca2+, and dithiothreitol. The acti vation was accompanied by the appearance of new sulfhydryl groups as d etermined with 5,5'-dithiobis(2-nitrobenzoic acid). By analyzing the a ctivated and nonactivated enzymes on SDS-polyacrylamide gel electropho resis under nonreducing conditions, it was found that an intermolecula r disulfide bridge between the small subunits of the potato tuber enzy me was reduced during the activation. Further experiments showed that the activation was mediated via a slow reduction and subsequent rapid conformational change induced by ADP-glucose, The activation process c ould be reversed by oxidation with 5,5'-dithiobis(2-nitrobenzoic acid) . Incubation with ADP-glucose and dithiothreitol could reactivate the oxidized enzyme. Chemical modification experiments with [C-14]iodoacet ic acid and 4-vinylpyridine determined that the intermolecular disulfi de bridge was located between Cys(12) of the small subunits of the pot ato tuber enzyme. Mutation of Cys(12) in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and pr evented the formation of the intermolecular disulfide of the potato tu ber enzyme, The mutants had instantaneous activation rates as the wild -type in the reduced state. A two-step activation model is proposed.