Affinity labeling of the allosteric activator site(s) of spinach leaf ADP-glucose pyrophosphorylase.
Pyridoxal-P has been shown to be an activator of the spinach leaf ADP-glucose pyrophosphorylase. It has a higher apparent affinity than the physiological activator 3-phosphoglycerate but only activates the enzyme activity 6-fold whereas 3-phosphoglycerate gives a 25-fold activation. Reductive phosphopyridoxylation of the spinach leaf enzyme results in enzyme having less dependence on the presence of activator for activity. Labeled pyridoxal-P is incorporated into both the 54- and 51-kilodalton subunits of the spinach leaf enzyme. The incorporation is inhibited by the presence of either 3-phosphoglycerate or the allosteric inhibitor, inorganic phosphate, thus suggesting that pyridoxal phosphate is covalently bound to the allosteric activator site. The pyridoxal phosphate is bound to an epsilon-amino group of a lysine residue. The phosphopyridoxylated enzyme is more resistant to phosphate inhibition than the unmodified form. The modified 51-kDa subunit has been digested with trypsin, and the peptide containing the labeled pyridoxal phosphate has been purified via high performance liquid chromatography and sequenced. Comparison of this sequence with the deduced amino acid sequence of a rice endosperm cDNA clone indicates that the putative allosteric site of the 51-kDa subunit is close to the carboxyl-terminal. This is in contrast to what had been demonstrated for the position of the activator site of the Escherichia coli ADP-glucose pyrophosphorylase which was shown to be close to the amino-terminal of the subunit.
Morell
M
M
Department of Biochemistry, Michigan State University, East Lansing 48824.
Bloom
M
M
Preiss
J
J
D000345:Affinity Labels / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004789:Enzyme Activation; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010449:Peptide Mapping; D010944:Plants / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology
D000345:Affinity Labels; D000596:Amino Acids; D011732:Pyridoxal Phosphate; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase