Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by CuII Terpyridine

Abstract

The hydrolysis and transesterification of RNA are catalyzed by a variety of metal ions, metal complexes, metalloenzymes, and ribozymes. These reactions are of fundamental biochemical importance. However, the role that metal ions play in RNA hydrolysis is not completely understood. We previously showed that aqueous Cu(II) terpyridine (Cutrpy) is effective for both transesterification and hydrolysis of RNA, and we harnessed this reactivity by constructing ribozyme mimics that employ terpyridyl Cu(II) in their active sites. Here we report a detailed kinetic study of the hydrolysis of adenosine-2‘,3‘-cyclic monophosphate (cAMP) by Cutrpy. The reaction is established to be first-order in Cutrpy and first-order in substrate. Catalytic turnover is observed, although product inhibition occurs. Chloride ion also inhibits the reaction, which indicates the disadvantage of using NaCl as an ionic strength buffer in related studies. The pH−rate profile is sigmoidal and implicates the hydroxide form of the catalyst, CutrpyOH+, as the active species. Isotope effects were used to determine whether the metal hydroxide acts as a nucleophile or a base. The solvent deuterium kinetic isotope effect, kH/kD, was measured to be 1.0 ± 0.1 after considering equilibrium isotope effects. To assist the mechanistic interpretation of the measured isotope effects, the fractionation factors for a dianionic phosphorane transition state (methyl phosphate dianion) and hydroxide were evaluated by ab initio quantum chemical calculations. Considering the calculated results along with the relevant experimental fractionation factors, isotope effects were predicted for three overall mechanisms: nucleophilic catalysis, general base catalysis, and specific base/general acid catalysis. We conclude that CutrpyOH+ acts as a nucleophilic catalyst in the hydrolysis of cAMP. This contrasts with the behavior of CutrpyOH+ as a base catalyst in the transesterification of RNA.