Biosynthesis of bacterial glycogen. Isolation and characterization of the pyridoxal-P allosteric activator site and the ADP-glucose-protected pyridoxal-P binding site of Escherichia coli B ADP-glucose synthase.
[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of the allosteric effector, 1,6-hexanediol-P2, protects against pyridoxylation of the allosteric activator site and allows modification of the ADP-glucose-protected site. The activator site CNBr [3H]pyridoxyl-P peptide was purified to homogeneity in the presence of urea by Sephadex G-50 and CM-cellulose chromatography. The peptide consists of 59 residues, with a molecular weight of 6750. The NH2-terminal of the peptide has a 16-residue sequence overlap with the previously determined NH2-terminal sequence of the native enzyme. The activator site pyridoxyl-P lysine is identified as residue 38 of the native enzyme's NH2 terminus. The ADP-glucose-protected site CNBr [3H]pyridoxyl peptide was purified to homogeneity by Sephadex G-50 and DEAE-cellulose chromatography. The peptide consists of 21 residues, with a molecular weight of 2460. The sequence of this peptide has been elucidated.
D000245:Adenosine Diphosphate Glucose / Q000378:metabolism*; D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005964:Glucosyltransferases / Q000378:metabolism*; D006003:Glycogen / Q000096:biosynthesis*; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D013214:Starch Synthase / Q000378:metabolism*
D000596:Amino Acids; D009702:Nucleoside Diphosphate Sugars; D010446:Peptide Fragments; D000245:Adenosine Diphosphate Glucose; D011732:Pyridoxal Phosphate; D006003:Glycogen; D005964:Glucosyltransferases; D013214:Starch Synthase