Biosynthesis of bacterial glycogen. Incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose-protected pyridoxal phosphate 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 allosteric effectors fructose-P2, 5'-AMP, or hexanediol-1,6-P2, protects against pyridoxylation of the allosteric activator site, and allows modification of the ADP-glucose-protected site. Incorporation of pyridoxal-P into the allosteric activator site results in modified enzyme of high activity form, even in the absence of fructose-P2. This modified enzyme, when assayed in the absence of fructose-P2, exhibits activation kinetics similar to nonpyridoxylated enzyme assayed in the presence of fructose-P2 and is still inhibited by 5'-AMP. These data suggest that the allosteric activator site of pyridoxylation is the fructose-P2 binding site, and is distinct from the inhibitor 5'-AMP binding site. Incorporation of pyridoxal-P into the ADP-glucose-protected site results in a decrease in enzyme activity. This pyridoxylated lysine could be involved with the binding of thesubstrates ADP-glucose, alpha-glucose-1-P, or PPi, or participate in the catalytic mechanism of the enzyme.
D000245:Adenosine Diphosphate Glucose / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D004926:Escherichia coli / Q000378:metabolism*; D006003:Glycogen / Q000096:biosynthesis*; D007700:Kinetics; D008024:Ligands; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*
D008024:Ligands; D009702:Nucleoside Diphosphate Sugars; D000245:Adenosine Diphosphate Glucose; D000249:Adenosine Monophosphate; D011732:Pyridoxal Phosphate; D006003:Glycogen; D009713:Nucleotidyltransferases