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 with 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 were replaced individually by an alanine residue, and the resulting enzymes were assayed as catalysts of poly(cytidylic acid) (poly(C)) cleavage. The values of Km and kcat/Km for poly(C) cleavage were affected only by replacing Arg-85. Moreover, the contribution of Arg-85 to the binding of the ground state and the transition state was uniform---Km increased by 15-fold and kcat/Km decreased by 10-fold. The contribution of Arg-85 to binding was also apparent in the values of Kd 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 interacts 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.