Why nature really chose phosphate

Abstract

Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

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.…”

“…[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. [40,54] Finally, the number of waters that might participate in proton transfer has also been a subject of debate. Thus, for computations that use PDB: 1wq1 as the initial model in QM/MM calculations, it has been argued that a critical proton transfer to substrate requires a second water molecule in addition to that which is the nucleophile in GTPase-catalyzed hydrolysis, even though this water is not seen in multiple high resolution MFx complexes (Section 8.5).…”

“…[20,40] In particular, Linear Free Energy Relationships (LFER) and Kinetic Isotope Effect (KIE) studies have supported a proposal that the hydrolysis of GTP in water is a dissociative process. [66] This analysis has been extrapolated to the Ras-catalyzed reaction, [67] with KIE measurements supporting PTx as proceeding via a loose TS in this enzyme.…”

“…Rather, these GSA and TSA structures have been widely used as starting points for a very large number of calculations by replacing the MFx moiety by PO3 while retaining the tbp geometry. The resulting structures have then permitted computations aimed at delineating the molecular mechanisms of a variety of enzymes catalyzing PTx reactions, [40] particularly the small GTPases, which play critical roles in cell signaling and regulation, and to cAPK. [41] Theoretical methods provide considerable insights into the distribution of electrons within molecules, and the energies of protein/ligand interactions that mediate binding and TS stabilization.…”

What can be learned about the enzyme ATPase from single-molecule studies of its subunit F1?

“…The F O F 1 -ATP synthase consists of the two units F O and F 1 but in the single molecule studies it is the F 1 part, which has been studied thus far. Since there is then no proton gradient across the F O ion channels to drive, via the c-ring and the γ shaft, the opening and closing of β subunits in the F 1 , almost all of the single molecule studies have been performed for the reverse reaction, namely the hydrolysis of ATP (Kamerlin et al 2013), catalyzed by the F 1 -ATPase. We note in passing that in some bacteria the F O F 1 -ATP synthase works in reverse, it hydrolyzes ATP to generate ion gradient and in vitro the enzyme can operate in both directions depending on the nucleotide and proton concentration (Pedersen & Carafoli, 1987).…”