The effect of magnesium ions on triphosphate hydrolysis

Abstract

Abstract:The role of metal ions in catalyzing phosphate ester hydrolysis has been the subject of much debate, both in terms of whether they change the transition state structure or mechanistic pathway. Understanding the impact of metal ions on these biologically critical reactions is central to improving our understanding of the role of metal ions in the numerous enzymes that facilitate them. In the present study, we have performed density functional theory studies of the mechanisms of methyl triphosphate and acetyl phosphate hydrolysis in aqueous solution to explore the competition between solvent-and substrate-assisted pathways, and examined the impact of Mg 2 + on the energetics and transition state geometries. In both cases, we observe a clear preference for a more dissociative solvent-assisted transition state, which is not significantly changed by coordination of Mg 2 + . The effect of Mg 2 + on the transition state geometries for the two pathways is minimal. While our calculations cannot rule out a substrate-assisted pathway as a possible solution for biological phosphate hydrolysis, they demonstrate that a significantly higher energy barrier needs to be overcome in the enzymatic reaction for this to be an energetically viable reaction pathway.

Challenges and advances in the computational modeling of biological phosphate hydrolysis

“…18,29,31 Our calculations suggest that for most phosphate monoester dianions, a solvent-assisted pathway dominates, although a substrate-assisted pathway becomes viable only for very poor leaving groups. 20 This is borne out also in our study of the effect of metal ions on phosphate hydrolysis, 25 where the solvent-assisted pathway is consistently energetically preferred, in line with our studies of phosphate monoesters. Finally, in the case of the pyridinio-N-phosphonates, 24 where proton transfer is no longer an issue, we obtain loose symmetrical transition states for the reaction with pyridine, in agreement with prior experimental work.…”

“…6. Our calculations 63 suggested that the phosphatase activity of this enzyme proceeds through a substrate-assisted mechanism, similar to that suggested for other phosphatases [34][35][36]91,92 (although this data will need revisiting in light of our more recent studies of model systems 20,25,63 ). In contrast, the sulfatase activity of this enzyme appears to utilize the active site histidine, H115, as a general base with which to deprotonate the active site nucleophile.…”

“…We will focus this section primarily on the hydrolysis of phosphate monoesters and related compounds, as these have been both extensively studied experimentally, 2,3 and also have been of particular interest to us in our recent work. 17,20,[23][24][25] Experimentally, the hydrolysis of phosphate monoesters is believed to proceed through loose, dissociative transition states, on the basis of the steep slope of the linear free energy relationship for the hydrolyses of these compounds (b lg = À1.23), 13 near-zero activation entropies, 26 as well as, in the case of p-nitrophenyl phosphate (pNPP) hydrolysis, kinetic isotope effects (KIE) 14,27 that show a large normal isotope effect on the bridging oxygen at the position of bond-cleavage (i.e., the oxygen in the axial position, bridging the phosphorus atom and the leaving group, O lg , 18 k bridge = 1.0189), an inverse isotope effect ( 18 k nonbridge = 0.9994) on the non-bridging oxygens (i.e., the oxygen atoms in the equatorial positions, O nb ), and a 15 k isotope effect that is close to the maximum value that would be expected for breaking the bond to the leaving group at the transition state ( 15 k = 1.0028). Computational studies, however, have been less conclusive, with both associative and dissociative transition states being proposed for the hydrolysis of these compounds, 18,[28][29][30][31] including an apparent leaving-group dependence for the preferred mechanism of hydrolysis.…”

“…Open Access Article. Based on this initial work, 17,23 we have addressed a range of related problems, including the effect of leaving group variation on phosphate monoester hydrolysis, 20 the effect of metal ions on the hydrolysis of methyl triphosphate and acetyl phosphate, 25 and the reactions of substituted pyridinio-Nphosphonates with pyridine, 24 which eliminates the challenges involved in addressing the competition between substrate-and solvent-assisted mechanisms. In the case of phosphate monoester hydrolysis with different leaving groups, we observe a leavinggroup dependent mechanistic shift, 20 in line with previous computational work.…”

Computer simulations of the catalytic mechanism of wild-type and mutant β-phosphoglucomutase

“…17,18 This method allows us to extensively sample the dynamics of each of these proteins in aqueous solution, and consistently compare the corresponding activation free energies with a single set of EVB parameters. We demonstrate that, in line with the corresponding reaction in aqueous solution, 19 there is a strong energetic preference for the D10-assisted pathway in the β-PGM active site. This mechanism is also the only mechanism that quantitatively reproduces changes in activity upon mutation of key active site residues.…”