Theoretical Study On The Hydrolysis Of Intracellular Second Messenger And Related Phosphodiesters

In this work, a series of theoretical methods were employed to investigate the none-enzymatic and enzymatic hydrolysis of intracellular second messenger adenosine 3', 5'-cyclic monophosphate (cAMP) and related phosphodiesters. The thesis consists of three chapters, including (1) theoretical study of none-enzymatic hydrolysis of cAMP, (2) theoretical determination of activation free energies for alkaline hydrolysis of cyclic and acyclic phosphodiesters in aqueous solution, and (3) theoretical study of the enzymatic hydrolysis of cAMP and related phosphodiester.For the studies described in the first chapter, .we have performed a series of first-principles electronic structure calculations to study competing reaction pathways and the corresponding free energy barriers for ester hydrolysis of 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 SN2 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 energetic 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 pK, of the ester and 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. When pH < ~ 3.7, the contribution of pathway (B) is larger. When 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 hydrolysis of TMP in solution.In the second chapater, first-principles electronic structure calculations were performed to examine the reaction pathway and corresponding activation free energies for alkaline hydrolysis of representative phosphodiesters, including dimethyl phosphate (DMP), trimethylene phosphate (TMP), ethylene phosphate (EP), and a simplified model (cAMPm) of adenosine 3', 5'-phosphate (cAMP). Reaction .coordinate calculations show that for all of these phosphodiesters the alkaline hydrolysis follows a one-step bimolecular mechanism initialized by the attack of hydroxide ion at thephosphorous atom of the ester. Five self-consistent reaction field (SCRF) methods were used to calculate the activation free energies and the calculated results were compared with available experimental data. It has been shown that the results calculated by using a recently developed SCRF method, known as the surface and volume polarization for electrostatics (SVPE) or fully polarizable continuum model (FPCM), which accurately determines both surface and volume polarization, are rather insensitive to the used solute charge isodensity contour value which determines the solute cavity size. The SVPE calculations plus nonelectrostatic interaction corrections led to activation free energies 32.6, 31.6, and 24.8 kcal/mol for DMP, TMP, and EP, respectively. The calculated activation free energies are all in good agreement with available experimentally estimated activation free energies -32, -32, and -21-24 kcal/mol for DMP, TMP, and EP, respectively. The FPCM results show that the solvation dramatically decreases the activation free energies for the alkaline hydrolysis of phosphodiesters...