1984 Volume 95 Issue 3 Pages 751-759
In order to clarify the subsite structure of ribonuclease A (RNase A), the interactions 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 P1 subsite. A similar 31P NMR titration was observed in the case of 5'-phosphate of pAp in the presence of RNase A. The results indicated that pAp interacted with RNase A at the P1, B2, and P2 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 P1 site of RNase A, thus indicating the binding of the 3'-side phosphates with the P2 subsite of RNase A.
The effects of pA, Ap, and adenosine on the RNase A-s4Up complex were studied by difference spectroscopy. The results indicated the binding of Ap at the B2 and P2 sites of RNase A without affecting the RNase A-s4Up complex. The rates of enzymatic cleavage of UpApA and UpApG were 2.47 and 5.17 times larger than that of UpA, respectively and were different from each other. This supports the existence of the B3 site besides the P2 site in the active site of RNase A. These results clearly indicated the presence of P2 and B3 sites in RNase A in addition to the P0, B1, P1, and B2 sites previously reported.
