Mononuclear and dinuclear mechanisms for catalysis of phosphodiester cleavage by alkaline earth metal ions in aqueous solution

In biological systems, enzymes often use metal ions, especially Mg2+, to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg2+ and Ca2+ catalyzed hydrolysis of the model phosphodiester thymidine-5′-p-nitrophenyl phosphate (T5PNP). At 25 °C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (kOH) of 4.1 × 10−4 M−1s−1 and 3.7 × 10−4 M−1s−1 in the presence of 0.30 M Mg2+ and Ca2+, respectively, compared to 8.3 × 10−7 M−1s−1 in the absence of divalent metal ion. Examining the dependence of kOH on [M2+] at 50 °C indicates different kinetic mechanisms with Mg2+ utilizing a single ion mechanism and Ca2+ operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the kOH values reveals a single Mg2+ catalyzes the reaction by 1800-fold whereas a single Ca2+ ion catalyzes the reaction by only 90-fold. The second Ca2+ provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg2+ and Ca2+.