Dinitrophenylation and inactivation of bovine pancreatic ribonuclease A

Abstract

The dinitrophenylation of bovine pancreatic ribonuclease A at pH 8.0, 15 °C is confined in its initial stages to the modification of lysine residues. The sites of attack are the α-amino group of the N-terminal lysine residue and the ϵ-amino groups of the lysine residues in positions 7 and 41. The most reactive amino group is that at position 41; it reacts with fluorodinitrobenzene about 70 times faster than the ϵ-amino group in glycyl- l-lysine. Substitutions at positions 1 and 41 appear to take place independently. However, examination of the product distribution reveals that substitution at position 7 is dependent on prior reaction at position 41. This implicates a rearrangement of the tertiary structure after reaction at that position. The 41-DNP-ribonuclease A is inactive, whereas modification of the α-amino group gives rise to a product with 60% of the specific activity of ribonuclease A. The rate of the inactivation reaction is strongly affected by competitive inhibitors of the enzyme, including ortho-phosphate ion, pyrimidine nucleosides, and both pyrimidine and purine nucleotides. The results are interpreted in terms of a hypothesis which regards the ϵ-amino group of the lysine residue in position 41 as associated with a positively charged site necessary for the binding of the anionic portion of the substrate and which provides a partial explanation for the enhanced reactivity of the amino group towards fluorodinitrobenzene.