The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis

Abstract

Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group pKa on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pKa increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pKas studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears.

The effect of magnesium ions on triphosphate hydrolysis

“…We previously suggested that in the absence of metal ions, the preferred pathway for the hydrolysis of phosphate monoesters with good leaving groups is one involving a loose, dissociative transition state [19,20]. We demonstrate that for both compounds studied here, the presence of magnesium ions increases the energetic difference between substrate-and solvent-assisted pathways, creating a greater preference for a solventassisted pathway than in the absence of the metal.…”

“…[7,8] and references cited therein). To partially address this issue, we recently performed detailed comparisons of a range of phosphate monoester dianions with different leaving groups, examining both leaving group effects and the effect of including explicit microsolvation in the calculations [19,20]. In addition to reasonable quantitative agreement between experimental and calculated activation free energies, our calculations reproduced both the linear free energy relationship for phosphate monoester dianion hydrolysis, as well as the experimentally observed kinetic isotope effects for p-nitrophenyl phosphate hydrolysis [19].…”

“…All transition states were obtained by structural perturbation of previously optimized transition state geometries for phosphate ester hydrolysis with eight explicit water molecules [19,20], with manual addition of magnesium ions and additional water molecules as relevant (see the previous paragraph for the number of water molecules included for each system), as well as partial 1-D scans of the P-O nuc distance to come closer to the saddle point where it was not possible to directly optimize new transition states. The resulting optimized transition states were characterized by means of frequency calculations as well as by following the intrinsic reaction coordinate (IRC) [45,46] as far as possible in both reactant and product directions, followed by unconstrained geometry optimizations at the same level of theory on the resulting structures to obtain optimized reactant and product states for each reaction pathway, thus verifying that we are examining the correct transition states for the processes of interest.…”

“…1) at the same level of theory, allowing for direct comparison between the different mechanistic options. We have also carried out the same level of calculations with an analogous study of acetyl phosphate (AcP) hydrolysis, again both with and without bound Mg 2 + , in order to be able to make a direct link to our previous studies of phosphate monoester dianion hydrolysis [19,20].…”

Theoretical Studies of the Catalytic Mechanism of the Dihydroxyacetone Kinase

“…In addition, DFT methods have been employed in many computational studies of phosphoryl transfer reactions in gas phase, with implicit solvent models or in the active site of enzymatic systems. 87,89,[160][161][162][163] Among the methods that follows this theory, the functional Becke three-parameter Lee-Yang-Parr (B3LYP) 94,95 has been the one selected to use in this PhD Thesis. This functional has been employed in a variety of systems, [164][165][166][167] including works of phosphate hydrolysis 81,107,168 and in phosphoryl transfer reactions in kinases 48,89,169 giving activation energies in agreement with results of experimental measurements.…”