Nucleotide binding and affinity labelling support the existence of the phosphate-binding subsite p2 in bovine pancreatic ribonuclease A

When the reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate was carried out in the presence of several natural mononucleotides, a decrease of 25-75% was found in the amount of the reaction product derivative II (the main product of the reaction which has the nucleotide label at the alpha-NH2 group of Lys-1). The efficiency of inhibition followed the order 3'-AMP greater than 5'CMP approximately equal to 5'AMP greater than 3'CMP. Previous studies indicate that this order reflects the extent of occupancy of p2, a phosphate-binding subsite adjacent to the catalytic centre. This finding suggests that derivative II is the result of affinity labelling and that the phosphate group of the halogenated nucleotide binds to p2 before the reaction takes place. The dissociation constants and stoichiometry of the interaction between native enzyme, derivative II and derivative E (homologous to derivative II, but labelled with a nucleoside instead of a nucleotide) with 3'AMP and 5'AMP at several pH values were also determined. Although in general one strong binding site was found, no strong binding occurs between 3'AMP and derivative II. It is concluded that the phosphate of the label occupies the same site p2, as the phosphate of 3'AMP. Finally, the pH dependence for the binding of 3'AMP and 5'AMP to RNAase A indicates that they bind to different protein groups. The results presented support the structure of the active site of ribonuclease A postulated previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571-579).