FIGURE 5.

Calculated pH dependence of the cleavage reaction of the hairpin ribozyme as a function of base pK_A, assuming general acid–base catalysis. The fractions of protonated acid f_A, unprotonated base f_B, and their product f_A·f_B have been calculated and plotted as a function of pH, following the approach of Bevilacqua (2003). The sections shaded in gray are the extreme values of pH not accessible to experimental study. Reaction rate should be proportional to the fraction of ribozyme in the appropriate state of protonation, i.e., f_A·f_B. Note that in these graphs f_A and f_B are plotted on a log₁₀ scale (left), while f_A·f_B is plotted on a linear scale (right). (A) Plot for pK_A values of 6 and 10 for the acid and the base, respectively. This corresponds to the natural ribozyme, assuming A38 is the acid and G8 is the base. Reaction rate increases with pH until reaching a plateau value close to neutrality. (B) Plot for pK_A values of 6 and 7.5 for the acid and the base, respectively. This corresponds to that expected for a ribozyme in which G8 has been substituted by a base with a pK_A of 7.5. A bell-shaped pH dependence is now expected. (C) Plot for pK_A values of 7.5 and 6 for the acid and the base, respectively. This corresponds to that expected for a ribozyme in which G8 has been substituted by a base with a pK_A of 7.5, now acting as the acid in the reverse reaction. Note that the shape of the pH profile is the same as that for the cleavage reaction.