A Thermodynamic Study of the Hydrolysis of Cytidine 2‘,3‘-Cyclic Phosphate

From the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (Received for publication, March 17, 1965) Bovine pancreatic ribonuclease catalyzes the hydrolysis of cytidine 2’,3’-cyclic phosphate according to the following equa- tion (1): Cytidine 2’ ,3’-cyclic phosphate + Hz0 --t cytidine 3’-phosphate (1) This reaction is generally considered to be a model for the second step of the biphasic hydrolysis of ribonucleic acid and conse- quently has been subjected to considerable scrutiny in recent years. A number of kinetic studies have been made of this reaction (2-5). The over-all reaction is generally stated to be “irreversible” (2, 3, 6, 7); however, no estimates of the equilib- rium constant have appeared in the literature. In fact, very little thermodynamic data related to cytidine 2’,3’-cyclic phos- phate and cytidine 3’-phosphate are available although the usefulness of such data is readily apparent. In the present study, the equilibrium constant of the hy- drolysis reaction has been determined by two independent methods. The first method utilizes the thermodynamic cycle librium constant utilizes radioactive tracers: is added to a solution of cytidine 3’-phosphate-%, and, after equilibrium has been est.ablished, the concentration of cytidine 2’,3’-cyclic phosphate-% present. is determined by the isotope dilution technique. From a knowledge of the equilibrium constant and the steady state kinetic parameters of the forward reaction some of the steady state kinetic parameters characterizing the reverse re- action can be calculated.