Covalent modification of the inhibitor-binding site(s) of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-AMP-incorporated peptides.
The photoaffinity inhibitor analog [2-3H]8-azido-AMP is specifically and covalently incorporated into Escherichia coli ADP-glucose synthetase. The reaction site(s) of [2-3H]8-azido-AMP with the enzyme was identified by reverse phase high performance liquid chromatography isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease-generated peptides containing the labeled analog. Three regions of modification, represented by six labeled peptides, accounted for over 85% of the covalently bound label. The major binding region of the azido analog, composed of residues 108-128, contained approximately 55% of the recovered covalently bound radioactivity. A single residue, Tyr-113, contained between 50 and 75% of the label found in the major binding region. This site is the same as the major binding region of the substrate site-specific probe, 8-azido-ADP-[14C]glucose (Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 1058-1064). Conformational analysis of this region predicts that it is a part of a Rossmann fold, the supersecondary structure found in many adenine nucleotide-binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido-AMP were also identified by chemical characterization. One region, containing 20% of the covalently bound label, was composed of residues 11-68. This region contains Lys-38, the previously determined pyridoxal phosphate-modified allosteric activator site (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). The third minor region of modification, residues 222-254, contained approximately 15% of the covalently bound label. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure.
D000245:Adenosine Diphosphate Glucose / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000201:enzymology*; D051599:Glucose-1-Phosphate Adenylyltransferase; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010777:Photochemistry; D011487:Protein Conformation
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; D000245:Adenosine Diphosphate Glucose; D000249:Adenosine Monophosphate; C021291:8-azidoadenosine 5'-monophosphate; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D003488:Cyanogen Bromide