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-beta-D-ribofuranosyl 5'-monophosphate was studied under several reaction conditions. The reaction, at pH 7.3, 40 degrees 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 alpha-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.