The Reaction of Bovine Pancreatic Ribonuclease A with 6‐Chloropurineriboside 5′‐Monophosphate: Evidence on the Existence of a Phosphate‐Binding Sub‐site

The chemical modification of bovine pancreatic ribonuclease A by 6‐chloropurine 9‐β‐d‐ribo‐furanosyl 5′‐monophosphate was studied under several reaction conditions. The reaction, at pH 7.3, 40°C and a nucleotide: enzyme molar ratio of 60, showed a high degree of specificity in comparison to those corresponding to the base or the nucleoside. The main derivative was isolated by means of CM‐cellulose chromatography. Subtilisin cleavage of this derivative showed that the substitution had taken place in the S‐peptide moiety. Tryptic digestion of the S‐peptide indicated that a lysine residue had been modified. Enzymatic and physico‐chemical considerations showed that the actual site of reaction was the α‐amino group of Lys‐1. The structural and kinetic properties of the derivative are consistent with the existence of a phosphate‐binding sub‐site near the N‐terminal region of the enzyme. Copyright © 1980, Wiley Blackwell. All rights reserved