Mechanism of action of polymeric aurintricarboxylic acid, a potent inhibitor of protein--nucleic acid interactions.
The mechanism of inhibition of protein--nucleic acid complex formation by polymeric aurintricarboxylic acid (ATA) was investigated by proton magnetic resonance spectroscopy. The approach was the synthesis of totally deuterated ATA, followed by a 100-MHz proton magnetic resonance study of its interaction with bovine pancreatic ribonuclease A (RNase), a model nucleic acid binding protein. The binding of ATA to RNase elicited chemical shift changes and line broadening in the C(2)--H resonances of histidyl residues 12 and 119, both of which are located in the active site, whereas that of histidyl residue 105, which resides on the exterior of the protein structure, is unaffected. (Histidyl residue 48 is not observed under our conditions except at high pH.) The epsilon-methylene protons of the lysyl side chains were also broadened upon the binding of ATA. Polymeric ATA displaces cytidine 2'-monophosphate and cytidine 3'-monophosphate from the active site of the enzyme as revealed by nuclear magnetic resonance spectroscopy. These observations suggest that the mechanism of action of ATA involves competition between the nucleic acid and the polymeric ATA for binding in the active site of the protein. Electron spin resonance spectroscopy reveals that polymeric ATA is a stable free radical, thus accounting for the major line broadening effect upon binding to protein. This finding may provide a powerful means of probing the nucleic acid binding site of proteins by proton magnetic resonance spectroscopy.
D000818:Animals; D001312:Aurintricarboxylic Acid / Q000494:pharmacology*; D001665:Binding Sites; D002417:Cattle; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010179:Pancreas / Q000201:enzymology; D011108:Polymers / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA; D012260:Ribonucleases / Q000378:metabolism*
D003509:Cyclohexanecarboxylic Acids; D011108:Polymers; D001312:Aurintricarboxylic Acid; D006639:Histidine; D012313:RNA; D012260:Ribonucleases