Open AccessProton‐Magnetic‐Resonance Studies of the Lysine Residues of Ribonuclease A
Author(s) -
BROWN Larry R.,
BRADBURY J. Howard
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb04131.x
Subject(s) - histidine , titration , chemistry , titration curve , lysine , bovine pancreatic ribonuclease , amino acid , ribonuclease , stereochemistry , rnase p , active site , resonance (particle physics) , crystallography , enzyme , biochemistry , organic chemistry , rna , physics , particle physics , gene
The amino groups of ribonuclease A (RNase‐A) have been methylated with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance (PMR) studies. Although enzymatic activity is lost, PMR difference spectroscopy and PMR studies of thermal denaturation show native conformation is largely preserved in methylated RNase‐A. Resonances corresponding to the NH 2 ‐terminal α‐amino and 10 ɛ‐amino N ‐methyl groups are titrated at 220 MHz to obtain p K values. After correction for the effects of methylation, using values previously derived from model compound studies, a p K of 6.6 is found for the α‐amino group, a p K of 8.6 for the ɛ‐amino group of lysine‐41 and p K values ranging from 10.6 to 11.2 for the other lysine ɛ‐amino groups. Interactions between lysine‐7 and lysine‐41 or between the α‐amino and ɛ‐amino groups of lysine‐1 have been proposed to account for deviations from simple titration behaviour. The correct continuities for the titration curves of the histidine H‐2 proton resonances have been confirmed by selective deuteration of the H‐2 protons. Titration curves for the H‐2 proton resonances of histidine‐12 and histidine‐119 of methylated RNase‐A show deviations from the titration curves for the native enzyme, indicating some alteration of the active‐site conformation. In the presence of phosphate, titration curves for the H‐2 proton resonances of histidine‐12 and histidine‐119 of methylated RNase‐A indicate binding of phosphate at the active site, but these curves continue to show deviations from the titration behaviour of native RNase‐A. The titration curve for the N ‐methyl resonance of lysine‐41 is perturbed considerably by the presence of phosphate, which indicates a possible catalytic role for lysine‐41.