Transition state stabilization by deaminases: Rates of nonenzymatic hydrolysis of adenosine and cytidine

Nonenzymatic rates of hydrolytic deamination of adenosine and cytidine by acids and bases analogous to side chains of naturally occurring amino acids are compared with the rates of uncatalyzed deamination in water and with the rates of the hydroxide- and hydrogen ion-catalyzed reactions. For adenosine, hydroxide ion is an effective catalyst, with a second-order rate constant of 7.5 × 10^?6 m^?1 s^?1 at 85°C and an energy of activation of 19.9 kcal/mol. Acid-catalyzed deamination of adenine proceeds with a second-order rate constant of 1.5 × 10^?6 m^?1 s^?1 at 85°C. At concentrations of 1 m and at pH values corresponding to their respective pK_a values, dimethylamine, acetate, selenide, imidazole, phosphate, and zinc(II) do not enhance the rate of deamination of adenosine beyond that observed in water, and 2-mercaptoethanol produces only a modest rate enhancement. The uncatalyzed rate of adenosine deamination in water is 8.6 × 10^?9 s^?1 at 85°C: extrapolation to 37°C and comparison with k_cat for rat hepatoma adenosine deaminase yield a rate enhancement by the enzyme of approximately 2 × 10¹²-fold. 1,6-Dimethyladenosine, the conjugate acid of which has a pK_a value much higher than that of adenosine, is not readily deaminated, suggesting that the uncatalyzed deamination of adenosine does not proceed by hydroxide ion attack on the rare protonated form of adenosine, but rather by attack on the neutral species. Deamination of cytidine is catalyzed most effectively by hydroxide ion, with a second-order rate constant of 4.5 × 10^?4 m^?1 s^?1 at 85°C and an energy of activation of 28.5 kcal/mol. The uncatalyzed rate of deamination of cytidine in water, which also exhibits an energy of activation of 28.5 kcal/mol, is 8.8 × 10^?8 s^?1 at 85°C. Comparison of the rate extrapolated to 25°C with k_cat for bacterial cytidine deaminase gives a rate enhancement for the enzyme of 4 × 10¹¹-fold. The C-5 proton of the pyrimidine ring of cytidine does not exchange with solvent during alkaline hydrolysis, suggesting that deamination under these conditions does not involve prior addition of water across the 5,6 double bond.