C-13 NMR INVESTIGATIONS ON RIBONUCLEASE-A

The proposed interaction between the amino acid residues Asp 14 and His 48 of ribonuclease A has been confirmed by 13C-NMR spectroscopy. The titration behaviour of the resonance of the side-chain carboxyl group of Asp 14 suggests a pKa of 6.5-7.0 for His 48. An equilibrium between different conformations of the enzyme has been observed which is associated with the deprotonation process of His 48. Upon this deprotonation a hydrogen bond between the side-chains of Asp 14 or His 48 and Tyr 25 seems to be formed as is suggested by the behaviour of a tyrosine Cζ resonance assigned to Tyr 25. One phenylalanine resonance broadens and moves upfield on the addition of the inhibitor Cyd-2′-P, being therefore assigned to Phe 120. The behaviour of this resonance suggests that the upfield shift results from the anisotropy of the cytidine ring. The three Phe signals are assigned to the three Phe residues. © 1979.