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

Abstract

In biological systems, enzymes often use metal ions, especially Mg(2+), to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg(2+) and Ca(2+) catalyzed hydrolysis of the model phosphodiester thymidine-5'-p-nitrophenyl phosphate (T5PNP). At 25 degrees 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 (k(OH)) of 4.1x10(-4)M(-1)s(-1) and 3.7x10(-4)M(-1)s(-1) in the presence of 0.30M Mg(2+) and Ca(2+), respectively, compared to 8.3x10(-7)M(-1)s(-1) in the absence of divalent metal ion. Examining the dependence of k(OH) on [M(2+)] at 50 degrees C indicates different kinetic mechanisms with Mg(2+) utilizing a single ion mechanism and Ca(2+) operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the k(OH) values reveals a single Mg(2+) catalyzes the reaction by 1800-fold whereas a single Ca(2+) ion catalyzes the reaction by only 90-fold. The second Ca(2+) provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg(2+) and Ca(2+).