Coulombic forces in protein-RNA interactions: Binding and cleavage by ribonuclease A and variants at Lys7, Arg10, and Lys66

The interactions between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extend beyond the scissile P-O-5' bond. Enzymic subsites interact with the bases and phosphoryl groups of the bound substrate. Those residues interacting with the phosphoryl group comprise the PO, P1, and P2 subsites, with the scissile bond residing in the P1 subsite. Here, the function of the PO and P2 subsites of RNase A is characterized in detail. Lys66 (PO subsite) and Lys7 and Arg10 (P2 subsite) were replaced with alanine residues. Wild-type RNase A and the K66A, K7A/R10A, and K7A/R10A/K66A variants were evaluated as catalysts for the cleavage of poly(cytidylic acid) [poly(C)] and for their abilities to bind to single-stranded DNA, a substrate analogue. The values of k(cat) and K-m for poly(C) cleavage were affected by altering the PO and P2 subsites. The k(cat)/K-m values for poly(C) cleavage by the K66A, K7A/R10A, and K7A/R10A/K66A variants were 3-fold, 60-fold, and 300-fold lower, respectively, than that of wild-type RNase A. These values indicate that the PO and P2 subsites contribute 0.70 and 2.46 kcal/mol, respectively, to transition-state binding. Binding experiments indicate that the PO and P2 subsites contribute 0.92 and 1.21 kcal/mol, respectively, to ground-state binding. Thus, the PO subsite makes a uniform contribution toward binding the ground state and the transition state, whereas the P2 subsite differentiates, binding more tightly to the transition state than to the ground state. In addition, nucleic acid binding to wild-type RNase A is strongly dependent on NaCl concentration, but this dependence is diminished upon alteration of the PO or P2 subsite. The logarithm of Kd is a Linear function of the logarithm of [Na+] over the range 0.018 M less than or equal to [Na+] less than or equal to 0.14 M, with partial derivative log K-d/partial derivative log [Na+] = 2.3 +/- 0.1, 1.8 +/- 0.1, 1.4 +/- 0.1, and 0.9 +/- 0.2 for nucleic acid binding to wild-type RNase A and the K66A, K7A/R10A, and K7A/R10A/K66A variants, respectively. Similar experiments with NaF and the wild-type enzyme yield a log K-d/partial derivative log [Na+] = 2.0 +/- 0.2, indicating that the anion makes only a small contribution to nucleic acid binding. Together these data provide a detailed picture of the contributions of Coulombic interactions to binding and catalysis by RNase A, and illuminate the general role of Coulombic forces between proteins and nucleic acids.