Investigation of nucleotide-ribonuclease-A complexes with high-resolution 31P-nuclear-magnetic-resonance spectroscopy.

Abstract

The chemical shift of the phosphorus resonance of various nucleotides has been studied as a function of pH in the absence and presence of ribonuclease A. The 31P‐signal of the nucleotides shifts upfield upon protonation of the phosphate moiety. Thus titration curves and pK values are obtained when the 31P chemical shift is plotted against the pH value. The titration curves are analyzed in terms of interactions of the phosphate group with the base or ribose moieties of the nucleotide itself or with amino‐acid side chains of the active site of ribonuclease A. The chemical shift of the phosphorus magnetic resonance of cytidine 2′‐phosphate when this nucleotide is bound to ribonuclease A resmebles that of the dianionic form of the free compound. Proton magnetic resonance studies indicated that the histidine residue 119 of ribonuclease A is bound to the phosphate group in enzyme‐nucleotide complexes [4]. This is now confirmed, since the apparent pK value of the phosphate group of these complexes is the same as the pK value of histidine‐119 determined by proton magnetic resonance spectroscopy. Possible arrangements of the hydrogen bonds between the imidazole group of histidine‐119 and the phosphate group are disscussed. From linewidth broadeining due to chemical exchange, an average life time of the complex of cytidine 2′‐phosphate with ribonuclease A is derived.