KINETIC AND EQUILIBRIUM STUDIES OF RIBONUCLEASE-CATALYZED HYDROLYSIS OF URIDINE 2',3'-CYCLIC PHOSPHATE

Initial velocity measurements were made of the ribonuclease-catalyzed hydrolysis of uridine 2′,3′-cyclic phosphate to 3′-uridine monophosphate in the pH range 4-8 at 25° in 0.1 m Tris-acetate-0.1 m NaCl. The pH dependence of the ratio of the turnover number to the Michaelis constant, kS/KS, for uridine 2′,3′-cyclic phosphate is essentially identical with that previously reported in the literature for cytidine 2′,3′-cyclic phosphate and for several dinucleoside phosphates. This indicates that the same ionizable groups on the enzyme are catalytically important for these substrates, and that the ionization state of the cytosine ring is unimportant in the enzyme-catalyzed hydrolysis of cytidine 2′,3′-cyclic phosphate. An ionizable group in its basic form and an ionizable group in its acid form with pK values of 5.4 and 6.4, respectively, are implicated in the enzymatic reaction. The apparent pK values associated with kS are 5.8 and 7.5. The equilibrium constant for the hydrolysis of uridine 2′,3′-cyclic phosphate to 3′-UMP at pH 5 and 25° was found to be 440 by an isotope dilution technique. This constant was used to calculate the turnover number for the reverse reaction as a function of pH. The mechanistic implications of these results are discussed. © 1969, American Chemical Society. All rights reserved.