Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP.
S-Adenosylhomocysteine hydrolase (AdoHcyase) has previously been identified as a cytoplasmic adenosine and cyclic AMP binding protein. In order to examine the relationship between the adenosine and cyclic AMP binding sites on this enzyme we have explored the use of 8-azido analogues of adenosine and cyclic AMP as photoaffinity reagents for covalently labelling AdoHcyase purified from human placenta. 8-Azidoadenosine (8-N3-Ado), like adenosine, inactivated AdoHcyase, and the rate of inactivation was greatly increased by periodate oxidation. In addition, 8-N3-Ado was found to participate in the first step in the catalytic mechanism for AdoHcyase, resulting in conversion of enzyme-bound NAD+ to NADH, although it was not a substrate for the full enzyme-catalysed reaction. Radioactively labelled 8-N3-Ado, its periodate-oxidized derivative and 8-azidoadenosine 3', 5'-phosphate (8-N3-cAMP) bound specifically to adenosine binding sites on AdoHcyase and, after irradiation, became covalently linked to the enzyme. Photoaffinity-labelled enzyme could be precipitated by monoclonal antibody to human AdoHcyase. Two observations suggested that cyclic AMP and adenosine bind to the same sites on AdoHcyase. First cyclic AMP and adenosine each blocked binding of both radioactively labelled 8-N3-Ado and 8-N3-cAMP, and second, digestion with V8 proteinase generated identical patterns of peptides from AdoHcyase that had been photolabelled with [32P]8-N3-cAMP and [3H]8-N3-Ado. Binding sites for cyclic AMP on AdoHcyase were found to differ functionally and structurally from cyclic AMP binding sites on the R1 regulatory subunit of cyclic AMP-dependent protein kinase.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D043383:Adenosylhomocysteinase; D000345:Affinity Labels / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D001665:Binding Sites; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D006801:Humans; D006867:Hydrolases / Q000037:antagonists & inhibitors*; D008024:Ligands; D010446:Peptide Fragments / Q000032:analysis; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D047428:Protein Kinase Inhibitors
D000345:Affinity Labels; D001386:Azides; D008024:Ligands; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; C017042:8-azidoadenosine-3',5'-monophosphate; C012760:8-azidoadenosine; D000242:Cyclic AMP; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine