title: Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5.
abstract: The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis*; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism; D001665:Binding Sites; D007202:Indicators and Reagents; D007700:Kinetics; D010782:Photolysis; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D000345:Affinity Labels; D001386:Azides; D007202:Indicators and Reagents; C047899:N-(4-azido-2-nitrophenyl)glycyl-(prolyl-prolyl-glycyl)(5); D011393:Procollagen-Proline Dioxygenase