Resolving Apparent Conflicts between Theoretical and Experimental Models of Phosphate Monoester Hydrolysis

Abstract

Understanding phosphoryl and sulfuryl transfer is central to many biochemical processes. However, despite decades of experimental and computational studies, a consensus concerning the precise mechanistic details of these reactions has yet to be reached. In this work we perform a detailed comparative theoretical study of the hydrolysis of p-nitrophenyl phosphate, methyl phosphate and p-nitrophenyl sulfate, all of which have served as key model systems for understanding phosphoryl and sulfuryl transfer reactions, respectively. We demonstrate the existence of energetically similar but mechanistically distinct possibilities for phosphate monoester hydrolysis. The calculated kinetic isotope effects for p-nitrophenyl phosphate provide a means to discriminate between substrate- and solvent-assisted pathways of phosphate monoester hydrolysis, and show that the solvent-assisted pathway dominates in solution. This preferred mechanism for p-nitrophenyl phosphate hydrolysis is difficult to find computationally due to the limitations of compressing multiple bonding changes onto a 2-dimensional energy surface. This problem is compounded by the need to include implicit solvation to at least microsolvate the system and stabilize the highly charged species. In contrast, methyl phosphate hydrolysis shows a preference for a substrate-assisted mechanism. For p-nitrophenyl sulfate hydrolysis there is only one viable reaction pathway, which is similar to the solvent-assisted pathway for phosphate hydrolysis, and the substrate-assisted pathway is not accessible. Overall, our results provide a unifying mechanistic framework that is consistent with the experimentally measured kinetic isotope effects and reconciles the discrepancies between theoretical and experimental models for these biochemically ubiquitous classes of reaction.

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].…”

“…2), such that it is either bridging the α-and β-phosphates, the β-and γ-phosphates or all three phosphates at once. As our previous work highlighted the importance of including explicit microsolvation when performing such calculations [19], we have included 8 and 14 water molecules in addition to the nucleophilic water molecule in our calculations of acetyl phosphate and methyl triphosphate, respectively, extending this to 15 and 17 water molecules, respectively in the presence of the magnesium ion in order to complete the solvation shell of the magnesium ion. The remainder of the solvent was described using an implicit solvent model.…”

“…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]. These calculations demonstrated a clear preference for a solventassisted pathway for phosphate monoesters with good leaving groups, while hinting at a potential transition to a substrate-assisted pathway for compounds with poor leaving groups, although this would only happen for leaving groups with quite high pK a s (previous work suggested a crossover at a leaving group pK a of ~ 13).…”

“…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].…”