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 dithiothreitol or by ATP, glucose- 1-phosphate, Ca2+, and dithiothreitol. The activation was accompanied by the appearance of new sulfhydryl groups as determined with 5, 5'-dithiobis(2-nitrobenzoic acid). By analyzing the activated and nonactivated enzymes on SDS-polyacrylamide gel electrophoresis under nonreducing conditions, it was found that an intermolecular disulfide bridge between the small subunits of the potato tuber enzyme 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 could 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 [14C]iodoacetic acid and 4-vinylpyridine determined that the intermolecular disulfide bridge was located between Cys12 of the small subunits of the potato tuber enzyme. Mutation of Cys12 in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and prevented the formation of the intermolecular disulfide of the potato tuber enzyme. The mutants had instantaneous activation rates as the wild-type in the reduced state. A two-step activation model is proposed.
Fu
Y
Y
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Ballicora
M A
MA
Leykam
J F
JF
Preiss
J
J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D017931:DNA Primers; D004229:Dithiothreitol / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D051599:Glucose-1-Phosphate Adenylyltransferase; D005958:Glucosephosphates / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D011198:Solanum tuberosum / Q000201:enzymology*; D013379:Substrate Specificity
D017931:DNA Primers; D005958:Glucosephosphates; D000255:Adenosine Triphosphate; C031590:glucose-1-phosphate; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D002118:Calcium; D004229:Dithiothreitol