Fundamental reaction pathways and free-energy barriers for ester hydrolysis of intracellular second-messenger 3′,5′-cyclic nucleotide

We have performed a series of first-principles electronic structure calculations to study competing reaction pathways and the corresponding free-energy barriers for the ester hydrolysis of intracellular second-messenger adenosine 3′,5′-cyclic monophosphate (cAMP) and related phosphodiesters including trimethylene phosphate (TMP). Reaction coordinate calculations show three fundamental reaction pathways for the ester hydrolysis, including (A) attack of a hydroxide ion at the P atom of the phosphate anion (an S _N2 process without a pentacoordinated phosphorus intermediate), (B) direct attack of a water molecule at the P atom of the anion (a three-step process), and (C) direct attack of a water molecule at the P atom of the neutral ester molecule (a two-step process). The calculated energy results show that for the reactions in the gas phase the free-energy barrier for pathway A is the highest and the barrier for the rate-controlling step of pathway C is the lowest. However, for the reactions in aqueous solution, the free-energy barrier calculated for pathway A becomes the lowest, and the two main hydrolysis pathways are A and B. We also have demonstrated how the pK _a of the ester and the pH of the reaction solution affect the relative contributions of different hydrolysis pathways to the total hydrolysis rate. Reaction pathway A should be dominant for the cAMP hydrolysis in neutral aqueous solution. However, the relative contribution of pathway A to the total hydrolysis rate should decrease with decreasing pH of the solution. For pH < ∼3.7, the contribution of pathway B is larger. For pH > ∼3.7, the contribution of pathway A is larger. The reliability of our theoretical predictions is supported by the excellent agreement of the calculated free-energy barrier with available experimental data for the hydrolysis of TMP in solution.