Toward the Design of Ribonuclease (RNase) Inhibitors: Ion Effects on the Thermodynamics of Binding of 2'-CMP to RNase A

Ribonucleases (RNases) possess a variety of biological activities and, under certain conditions, are deleterious. Hence, design of selective inhibitors has been suggested as a strategy for treating RNase-related disorders. In the present study, isothermal titration calorimetry was used to measure ion effects on binding thermodynamics of the RNase A competitive inhibitor 2'-CMP as a representative system. The reaction cell (37degreesC) contained dialyzed RNase A (0.04-0.05 mM) in buffered solution (pH 5.5) of 50 mM Na+,K+,Ca2+, or Mg2+ acetate, verified spectrophotometrically. Thirty-five sequential injections (4 mul each, 3 min apart) were made of 2'-CMP (1.2 mM) in ion-matching buffer. The data were corrected for heat of dilution. There was a 1: 1 interaction in each case. The estimated parameters (+/-S.D.) were: K-d=4.84+/-0.29 muM (Na+); 5.62+/-0.98 muM (K+); 24.44+/-6.96 muM (Ca2+); 28.74+/-0.43 muM (Mg2+); DeltaG(o) = -7.541+/-0.037 kcal/mol (Na+); -7.458+/-1.03 kcal/mol (K+); -6.574+/-0.173 kcal/mol (Ca2+); -6.442+/-0.009 kcal/mol (Mg2+); DeltaH(o) = -22.357+/-1.189 kcal/mol (Na+); -21.917+/-0.891 kcal/mol (K+); -20.223+/-1.503 kcal/mol (Ca2+); -26.570+/-1.579 kcal/mol (Mg2+); and DeltaS(o) = -0.048+/-0.004 kcal/mol-K (Na+); -0.047+/-0.003 kcal/mol-K (K+); -0.044+/-0.005 kcal/mol-K (Ca2+); -0.065+/-0.005 kcal/mol-K (Mg2+). Thus, all reactions were enthalpy-driven. Despite a 5-fold difference in K-d between mono- and divalent ions, the ratio of ion hydration DeltaG(o) to K-d was constant. These data should be useful for molecular modeling and suggest that inhibitor activity will be a function of cellular conditions (normal or pathological).