The Journal of Biological Chemistry

The interaction of ribonuclease A with cupric and zinc ions has been studied spectrophotometrically and by potentiometric titration in the presence and absence of cytidylic acid derivatives. In the absence of cytidylic acid, the results suggest that each cupric ion distributes among a set of approximately four spectrally similar sites; each site appears to consist mainly of a single imidazole side chain and its adjacent peptide bond nitrogen atoms at neutral pH. No evidence for the preferential chelation of a single cupric or zinc ion between histidines-12 and -119 has been found. In the presence of 3’-cytidylate, binding of both cupric and zinc ions is strengthened. In addition, spectrophotometric studies indicate that cupric ion-binding sites are altered in the presence of 3’qtidylate. These data are compatible with formation of a ternary complex between a cupric or zinc ion, 3’-cytidylate, and ribonuclease. By contrast, 2’-cytidylate does not alter the type of ligand to which cupric ion binds, but appears to diminish very slightly the affinity of ribonuclease for cupric ion; these results suggest that 2’cytidylate may compete for one of the sites usually available to cupric ion. Several possible mechanisms for the inhibition of ribonuclease by cupric and zinc ions are suggested. These include blocking of the active site by bound metal ions and conformational changes induced by binding of metal ions elsewhere in the protein. The importance of cooperative interactions between 3’-cytidylate and metal ions in binding to ribonuclease is particularly stressed as a mechanism of inhibition.