Some Evidence Suggesting the Existence of P2 and B3 Sites in the Active Site of Bovine Pancreatic Ribonuclease A1

In order to clarify the subsite structure of ribonuclease A (RNase A), the interac-tions of pdTp, pAp, dTpdAp, and pdTpdAp with RNase A were investigated by means of kinetic studies and 31P NMR spectroscopy. The pH profile of the 31P NMR spectrum of RNase A-pdTp complex indicated the interaction of the 3'- and 5'-phosphates with RNase A. The signal of 3'-phosphate of pdTp in the presence of RNase A gave a characteristic titration curve indicating the participation of more than 2 ionic groups in the Px subsite. A similar 31P NMR titration was observed in the case of 5'-phosphate of pAp in the presence of RNase A. The results indi-cated that pAp interacted with RNase A at the Plf B,, and P, sites. dTpdAp and pdTpdAp inhibited RNase A action more markedly than dTpdA, indicating the contribution of 3'- and 5'-terminal phosphate groups attached to dTpdA to the affinity of RNase A. The 31P NMR spectra of RNase A-dTpdAp and pdTpdAp complexes excluded the possible interaction of the monoester type phosphate of the inhibitors with the Px site of RNase A, thus indicating the binding of the 3'-side phosphates with the P, subsite of RNase A