The Role of Lysine-41 in Ribonuclease A Studied by Proton-Magnetic-Resonance Spectroscopy of Guanidinated Ribonuclease A

Abstract

RNase A (EC 3.1.4.22) was guanidinated at the lysine residues and the nona-guanidinated and deca-guanidinated (fully substituted) products separated. In confirmation of an earlier report by Glick and Barnard, it was shown by chemical procedures that the former derivative is not reacted at lysine-41. Guanidination of lysine-41 to produce the fully substituted product causes loss of enzymic activity without any apparent change of conformation, as tested by conformational comparisons (using PMR spectroscopy) including difference spectroscopy, heat denaturation and titration of the active-site histidine residues 12 and 119. This is good evidence for the involvement of lysine-41 in a catalytic role in the enzyme. Dimethylation of lysine-41 of nona-guanidinated RNase A produces sharp proton resonances which shift as the dimethylamino group is titrated and allow the determination of an apparent pK of 8.8 for unsubstituted lysine-41.