Calorimetric evidence for conformational transitions of RNase A in the presence of cytidine 2′,3′-cyclic phosphate

Thermal denaturation of ribonuclease A (RNase A) complex with cytidine 3′-monophosphate (3′CMP) was studied by differential scanning calorimetry (DSC). The kinetic and binding studies of RNase A with cytidine 2′,3′-cyclic phosphate (cCMP) as a substrate, and 3′CMP as a ligand were also investigated by difference spectrophotometry. The obtained kinetic saturation curve reveals the occurrence of an anomalous non-hyperbolic shape at high substrate concentrations, and a biphasic binding isotherm. These phenomena indicate that a conformational change is occurring with RNase A during the hydrolysis of cCMP. A combination of kinetic and thermodynamic studies tends to elucidate the reasons for the formation of a non-hyperbolic behavior in a kinetic saturation curve. The thermal profile of the enzyme-3′CMP complexes shows a splitting of two distinct peaks with different structural stabilities of melting points (Tm) of 325 and 337 K. The bifurcate appearance of DSC profile of RNase A-3′CMP complexes manifests a physical view of a light kinetic structural transition. It is worthy to note, the direct binding (not via enzymatic reaction) of enzyme with 3′CMP indicates single DSC profile and monophasic binding isotherm.