Addressing Open Questions about Phosphate Hydrolysis Pathways by Careful Free Energy Mapping

Abstract

The nature and mechanism of phosphate hydrolysis reactions are of great interest in view of the crucial role of these reactions in key biological processes. While it is becoming clearer that the ultimate way of resolving mechanistic controversies must involve reliable theoretical studies, it is not widely realized that such studies cannot be performed by using most existing automated ways and that only careful systematic mapping approach can lead to meaningful conclusions. The present work clarifies the above point by considering the hydrolysis of phosphate monoesters. The clarification starts by defining the actual issues that should be addressed in careful studies, and by highlighting the problems with studies that ignore the need for unique mechanistic definitions (e.g., works that confuse associative and dissociative pathways). We then focus on the analysis of the proton transfer (PT) pathways in phosphate hydrolysis and on recent suggestions that PT involves more than one water molecule. Here we point out that most of the studies that found a proton transfer through several water molecules have not involved a systematic search of the relevant reaction coordinates. This includes both energy minimization approaches as well as a recent metadynamics (MTD) simulation study. To illustrate the crucial need of exploring the potential surfaces reliably, rather than relying on automated approaches, we present here a very careful study of the free energy landscape along a 3D reaction coordinate (RC) exploring both the standard 2D RC, comprised of the attacking and leaving group reaction coordinates, as well as of the proton transfer (PT) coordinate. Our study points out that that QM/MM minimization or MTD studies, that concluded that the hydrolysis of phosphate monoesters involves a PT through several water molecules, have not explored the single water (1W) path (that involves a direct PT form the attacking water molecule to the phosphate oxygen). Furthermore, we identified the most likely reason for the difficulty in finding the 1W path by QM/MM minimization methods, as well as by the current MTD simulations. We also discuss the problems with current studies that challenge the phosphate as a base mechanism. Perhaps most importantly, we clarify and illustrate that crucial mechanistic problems with alternative pathways should not be explored by running black box search approaches.

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

“…2). [40,49,52] On the other hand, other workers have reported a variety of QM and QM/MM studies in which they present evidence for a loose (more dissociative) TS (Scheme 1). [43,53] Similarly, there is substantial disagreement about the true functional role of a conserved active site glutamine, particularly regarding whether it mediates proton transfer.…”

“…[55] However, the energetic penalty for introducing this "second" water is estimated to be within thermal energy. [56] While such disparate conclusions may reflect inherent differences in the computational methods chosen to model reaction mechanism, and the inclusion or absence of adequate conformational sampling, it is also possible that the quality of the MFx-containing crystal structure might influence the calculations, especially if extensive equilibration using dynamics is not performed as part of geometry optimization and locating the TS. [49] As we point out above, there is considerable variation in the quality of MFx structures deposited in the PDB.…”

“…[68] Similarly, QM studies have invoked a second water molecule to assist in proton transfer in the TS for hydrolysis in aqueous solution. [52,56] This proposition has been developed into a "two water" mechanism for enzymatic hydrolysis of GTP based on a structure for Ras at 2.5 Å resolution (PDB: 1wq1) which has a less well-defined assembly of residues involved in the TS. [69] What is the evidence for these proposals from MFx studies?…”

Why nature really chose phosphate

“…One of the interesting insights that emerged very recently form of examination of the two water (2 W) versus 1 W mechanism (Prasad et al 2012) is the finding (see Figs 21 and 22) that the compression of R2 (the distance between the oxygen of the attacking water and the phosphate) drastically changes the pK a of the attacking water (from about 16 to a negative value). Obviously, at some stage the attacking water becomes very acidic and transfers a proton either to another water or to the phosphate oxygen.…”

“…It is also worth pointing out that although the studies of (Grigorenko et al 2005(Grigorenko et al , 2006 were instructive (in particular for providing new insight into the initial orientation of the nucleophile), they are also problematic for a number of reasons. These studies never examined the full surface, and have not used any sampling (the corresponding problems with which have been specifically demonstrated in Klähn et al 2006) and now in Figs 19 and 20 (Prasad et al 2012). Thus, the authors arrived at a questionable mechanism in the Ras-GAP complex, for which the first step (P-O bond cleavage) had a barrier of only about 4 kcal mol x1 (Grigorenko et al 2007a), where a complex of water and metaphosphate is being formed, with a subsequent rate-determining barrier of 10Á5 kcal mol x1 relative to the reactant state, leading to Gln-assisted conversion of the nucleophilic water molecule plus metaphosphate to Pi+GDP with a protonated Gln sidechain in the enzyme.…”