1H-n.m.r. studies on the specificity of the interaction between bovine pancreatic ribonuclease A and dideoxynucleoside monophosphates

The titration curves of the C-2 histidine protons of bovine pancreatic ribonuclease A in the presence of several dideoxynucleoside monophosphates (dNpdN) were studied by means of proton nuclear magnetic resonance at 270 MHz in order to obtain information on the ligand — RNase A interaction. The changes in the chemical shift and pKs of the C-2 proton resonances of His-12, -48, -119 in the complexes RNase A — dNpdN were smaller than those previously found when the enzyme interacted with mononucleotides. The pK₂ of His-12 was not affected by the interaction of the enzyme with these ligands, whereas, the perturbation of the pK₂ of His-119 was clearly dependent on the nature of the ligand. If there is a pyrimidine nucleoside at the 3′ side of the dideoxynucleoside monophosphates, as in TpdA and TpT, an enhancement due to the well known interaction of the phosphate in p₁, the catalytic site, was found. However, when there is a purine nucleoside, as in dApT and dApdA, a decrease in the pK₂ value was observed and we propose that in such cases the phosphate group interacts in a secondary phosphate binding site, p₂. The results obtained suggest the existence of different specific interactions depending on the structure of the dideoxynucleoside monophosphate studied.