Open AccessNuclear‐Magnetic‐Resonance Study of the Histidine Residues of S‐Peptide and S‐Protein and Kinetics of 1 H‐ 2 H Exchange of Ribonuclease A
Author(s) -
BRADBURY J. Howard,
CROMPTON Malcolm W.,
TEH Joo Seng
Publication year - 1977
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1977.tb11966.x
Subject(s) - histidine , chemistry , bovine pancreatic ribonuclease , reaction rate constant , rnase p , titration , resonance (particle physics) , hydrogen–deuterium exchange , crystallography , ribonuclease , solvent , nuclear magnetic resonance spectroscopy , kinetics , chemical shift , analytical chemistry (journal) , stereochemistry , hydrogen , enzyme , chromatography , organic chemistry , biochemistry , atomic physics , quantum mechanics , rna , physics , gene
1 H NMR spectroscopy at 100 MHz was used to determine the first‐order rate constants for the 1 H‐ 2 H exchange of the H‐2 histidine resonances of RNase‐A in 2 H 2 O at 35°C and pH meter readings of 7, 9, 10 and 10.5. Prolonged exposure in 2 H 2 O at 35°C and pH meter reading 11 caused irreversible denaturation of RNase‐A. The rate constants at pH 7 and 9 agreed reasonably well with those obtained in 1 H‐ 3 H exchange experiments by Ohe, J., Matsuo, H., Sakiyama, F. and Narita, K. [ J. Biochem. (Tokyo) 75 , 1197–1200 1974)]. The rate data obtained by various authors is summarised and the reasons for the poor agreement between the data is discussed. The first‐order rate constant for the exchange of His‐48 increases rapidly from near zero at pH 9 (due to its inaccessibility to solvent) with increase of pH to 10.5. The corresponding values for His‐119 show a decrease and those for His‐12 a small increase over the same pH range. These changes are attributed to a conformational change in the hinge region of RNase‐A (probably due to the titration of Tyr‐25) which allows His‐48 to become accessible to solvent. 1 H NMR spectra of S‐protein and S‐peptide, and of material partially deuterated at the C‐2 positions of the histidine residues confirm the reassignment of the histidine resonances of RNase‐A [Bradbury, J. H. & Teh, J. S. (1975) Chem. Commun. , 936–937]. The chemical shifts of the C‐2 and C‐4 protons of histidine‐12 of S‐peptide are followed as a function of pH and a p K ′ value of 6.75 is obtained. The reassignment of the three C‐2 histidine resonances of S‐protein is confirmed by partial deuteration studies. The p K ′ values obtained from titration of the H‐2 resonances of His‐48, His‐105 and His‐119 are 5.3, 6.5 and 6.0, respectively. The S‐protein is less stable to acid than RNase‐A since the former, but not the latter, shows evidence of reversible denaturation at pH 3 and 26°C. His‐48 in S‐protein titrates normally and has a lower p K than in RNase‐A probably because of the absence of Asp‐14, which in RNase‐A forms a hydrogen bond with His‐48 and causes it to be inaccessible to solvent, at pH values below 9.