Comparison of free vs. immobilized RNase-A kinetics and binding thermodynamics of a pyrimidine nucleotide using isothermal titration calorimetry

The present study used isothermal titration calorimetry (ITC) to compare the equilibrium thermodynamics and kinetics of an immobilized enzyme vs. the same enzyme free in solution. ITC is an ultra-sensitive method for monitoring changes in temperature of biochemical interactions and the thermodynamics and affinity of chemical reactions. Bovine pancreatic ribonuclease (RNase-A) was chosen based on: (1) its well-characterized reaction scheme, (2) its availability in sufficient quantities, and (3) possible implications for clinical application. Specifically, ITC was used to compare the kinetics of the conversion of substrate 2'3'-cytidine monophosphate (cCMP) to product, cytidine 3'-monophosphate (3'CMP) by RNase-A free in solution vs. immobilized on a polymer bead complex (Sepharose 4bRTM), and the equilibrium thermodynamics of the binding of the RNase-A competitive inhibitor cytidine 2'-monophosphate (2'CMP). From integrated calorimetric data, the parameters DeltaHobs, DeltaG° and DeltaS° and the dissociation constant Kd were obtained for comparison at 298, 304 and 310° K (pH 5.5). From differential calorimetric data, the Michaelis-Menten based parameters Km, kcat and Kp (end-product dissociation constant) were obtained at 298 and 310°K (pH 5.5). Overall comparison of the immobilized to free RNase-A/cCMP kinetic parameters revealed: (1) a decrease in turnover rate (kcat), (2) weaker substrate binding (Km), and (3) increase in end-product affinity (Kp). Overall comparison of the immobilized to free RNase-A/2'CMP thermodynamics revealed: (1) a decrease in enthalpy and entropy change at 298° K (DeltaDeltaHobs = -5 kcal/mole; DeltaDeltaS° = -15.16 cal/mole-K), (2) an increase in enthalpy and entropy change at 310°K (DeltaDeltaHobs = 6.3 kcal/mole; DeltaDeltaS° = 21.16 cal/mole-K), (3) no change at 304°K, (4) an increase in 2'CMP binding affinity (Kd) at all three temperatures, and for immobilized RNase-A (5) no change in reaction enthalpy with temperature. These results give insight into the changes associated with immobilization of an enzyme and further demonstrate the utility of using ITC to measure kinetic and thermodynamic parameters of enzymes.