Covalent modification of the inhibitor binding site(s) of Escherichia coli ADP-glucose synthetase: specific incorporation of the photoaffinity analogue 8-azidoadenosine 5'-monophosphate.
The photoaffinity agent 8-azidoadenosine 5'-monophosphate (8-N3AMP) is an inhibitor site specific probe of the Escherichia coli ADP-glucose synthetase (ADPG synthetase). In the absence of light, 8-N3AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), the analogue specifically and covalently modifies the enzyme, and photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADPG provides nearly 100% protection from 8-N3AMP photoinactivation, while the substrate ATP provides approximately 50% protection and the inhibitor AMP, approximately 30% protection. These three adenylate allosteric effectors of E. coli ADPG synthetase also protect it from photoincorporation of 8-N3AMP. A structural overlap of the inhibitor and substrate binding sites is proposed which explains the protection data in light of the known binding and kinetic properties of this tetrameric enzyme.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000494:Allosteric Regulation; D001386:Azides / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors*; D010782:Photolysis
D000345:Affinity Labels; D001386:Azides; D000249:Adenosine Monophosphate; C021291:8-azidoadenosine 5'-monophosphate; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase