Product Inhibition and the pH Dependence of the Reverse Reaction Velocity of Ribonuclease A

The inhibition of ribonuclease by cytidine 3'-phosphate, a reaction product, was investigated from pH 6.3 to pH 8.3. Rates of cytidine 2',3'-cyclic phosphate hydrolysis were determined at different product concentrations, and the results were analyzed by a novel procedure, the linear plot of (S)0/v against (P)0 at a particular pH and constant (S)0. The slope of this plot, corrected to provide for the ionization state of the product, is inversely proportional to the maximal velocity of the reverse reaction. The symmetry of the plot of maximal velocity against pH for the reverse reaction suggests that the velocity of the reverse acidic pathway is equal to the velocity of the reverse alkaline pathway. Apparent pK values of pH 6.95 and 7.59 were calculated for the acidic dissociations of the cytidine 3'-phosphate ribonuclease complexes at an ionic strength of 0.01. The pK values of the product-enzyme complexes, however, are shifted to a lower pH at an ionic strength of 0.15. A tentative explanation of the shift to acidic pH at high ionic strength is the formation of ternary complexes between the divalent anion form of cytidine 3'-phosphate, ribonuclease, and the cationic component of the buffer.