A new remote subsite in ribonuclease A

The interaction between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extends beyond the scissile bond. Enzymic subsites interact w ith the bases and the phosphoryl groups of a bound substrate. We evaluated the four cationic residues closest to known subsites for their abilities to interact with a bound nucleic acid. Lys-37, Arg-39, Arg-85, and Lys-104 we re replaced individually by an alanine residue, and the resulting enzymes w ere assayed as catalysts of poly(cytidylic acid) (poly(C)) cleavage. The va lues of K-m and k(cat)/K-m for poly(C) cleavage were affected only by repla cing Arg-85. Moreover, the contribution of Arg-85 to the binding of the gro und state and the transition state was uniform-K-m increased by 15-fold and k(cat)/K-m decreased by 10-fold, The contribution of Arg-85 to binding was also apparent in the values of K-d for complexes with oligonucleotides of different length. This contribution was dependent on salt concentration, as expected from a coulombic interaction between a cationic side chain and an anionic phosphoryl group. Together, these data indicate that Arg-85 intera cts with a particular phosphoryl group of a bound nucleic acid. We propose that Arg-85 comprises a new distal subsite in RNase A-the P(-1) subsite.