Subsite interactions of ribonuclease T1: binding studies of dimeric substrate analogues

Ultraviolet difference spectral binding studies of ribonuclease T1 with pGp, ApG, CpG, UpG, DGpdA, dGpdC, dGpdG, dGpdT, dTpdG, pdApdG, pdTpdG, pdGpdA, pdGpdG, pdGpdT, c(pdGpdA), and c(pdGpdG) were conducted at pH 5.0, 0.2 M ionic strength and 25 degrees C. Under these conditions, the characteristic difference spectrum and association constant for (1:1) ribonuclease T1 binding were determined for each ligand. The binding of guanosine and deoxyguanosine containing ligands could be distinguished by the shapes of their difference spectra. The results indicated that the guanine moiety of each ligand was bound at the enzyme's primary recognition site. Evidence of a specific enzyme subsite for binding the adenine moiety of ApG and pdApdG is presented. The proposal of a specific enzyme subsite for binding the 5'-phosphate group of a complexed guanosine moiety (Sawada, F., Samejima, T., and Saneyoshi, M. (1973), Biochim. Biophys. Acta 299, 596) is not supported in the present work. Preliminary evidence for the existence of two additional enzyme subsites and the effect of oligomer conformation on enzyme binding are also discussed.