A mechanism of catalyzed GTP hydrolysis by Ras protein through magnesium ion

Lu, Qiang; Nassar, Nicolas; Wang, Jin

doi:10.1016/j.cplett.2011.09.071

Cited by 9 publications

(9 citation statements)

References 34 publications

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“…They range from irrelevant gas-phase calculations, to even recent puzzling QM/MM results providing a barrier of 70 kcal mol x1 for a completely impossible mechanism (Lu et al 2011), to a decent ab initio QM/MM study of phosphate hydrolysis in solution (Wenjin et al 2012) mentioned above. Presently, it would seem that the most effective way forward has been to study the solution reaction by an implicit solvation model (e.g.…”

Section: Mechanistic Insights Into Phosphate Hydrolysis In Proteinsmentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

335

448

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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.

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Section: Mechanistic Insights Into Phosphate Hydrolysis In Proteinsmentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

335

448

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“…Furthermore, some calculated activation energies deviate largely from experimental values. In different simulations, the energy barriers for GTP hydrolysis in Ras•GAP vary among 14 kcal∕mol (1 kcal ¼ 4.18 kJ) (14,17), 21 kcal∕mol (13,22), and up to 70 kcal∕mol (21). Of these simulations, only 14 kcal∕mol (14, 17) seems reasonably close to the experimental value and therefore appears to be the most appropriate.…”

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confidence: 99%

“…FTIR investigations thereby provide detailed insights into the molecular reaction mechanism (36, 37). Previous QM/MM simulations of GTP bound to Ras (16) and Ras•GAP (19), dealing with structural alterations, lacked the contribution of the magnesium ion (Mg 2þ ), which is important for the catalytic reaction (15,21,38). Here, in addition to the triphosphate itself, Mg 2þ , its coordinating partners, and the ribose are treated quantum mechanically.…”

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confidence: 99%

“…This is effected by GTPase-activating proteins (GAPs) that interact with Ras (6). The mechanisms of GTP hydrolysis have been extensively investigated both theoretically (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) and experimentally by X-ray crystallography (23)(24)(25)(26)(27), electron spin resonance (28)(29)(30), and FTIR spectroscopy (5,6,(31)(32)(33).…”

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confidence: 99%

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Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

Rudack

Xia

Schlitter

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

102

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Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and P i by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 10 5 compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 10 5 compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with timeresolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ-relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α-relative to the β-and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.enzyme catalysis | reaction mechanism | free energy of activation M any cellular processes are regulated by members of the Ras superfamily. They are switched "on" by GDP-to-GTP exchange and "off" by a GTPase reaction. GTP hydrolysis is vitally important for the regulation of several signal-transduction processes in living cells (1, 2). In the case of Ras, external growth signals are transduced to the nucleus. Site-specific oncogenic mutations inhibit the GTPase reaction and the growth signal can no longer be down-regulated. This contributes to uncontrolled cell growth, eventually leading to cancer (3). Common to all members of the Ras superfamily is the catalysis of GTP hydrolysis by the G domain (4). Ras-catalyzed GTP hydrolysis is five orders of magnitude faster than in water (30 min vs. 200 d) (5). However, to control growth signals in the living cell, a further increase of five orders of magnitude in the reaction rate (30 min to 50 ms) is enabled. This is effected by GTPase-activating proteins (GAPs) that interact with Ras (6). The mechanisms of GTP hydrolysis have been extensively investigated both theoretically (7-22) and experimentally by X-ray crystallography (23-27), electron spin resonance (28-30), and FTIR spectroscopy (5,6,(31)(32)(33).Determination of the three-dimensional structures of Ras and GAP by X-ray crystallography represents an important milestone in the understanding of t...

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“…[8,23] A series of recent reports by Gerwert and colleagues [23,68,86] have provided arguably some of the most detailed insights into the role of Mg 2+ in Ras- and Ras·GAP-catalysed GTP hydrolysis reactions. (Although their work goes far beyond the role of Mg 2+ and spans many years, [18–20] here we limit our discussion to the most recent ones that highlight the role of Mg 2+ .)…”

Section: Key Players In the Ras-catalysed Gtp Hydrolysis Reactionmentioning

confidence: 99%

Overview of simulation studies on the enzymatic activity and conformational dynamics of the GTPase Ras

Prakash

Gorfe

2014

Molecular Simulation

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Over the last 40 years, we have learnt a great deal about the Ras onco-proteins. These intracellular molecular switches are essential for the function of a variety of physiological processes, including signal transduction cascades responsible for cell growth and proliferation. Molecular simulations and free energy calculations have played an essential role in elucidating the conformational dynamics and energetics underlying the GTP hydrolysis reaction catalysed by Ras. Here we present an overview of the main lessons from molecular simulations on the GTPase reaction and conformational dynamics of this important anti-cancer drug target. In the first part, we summarise insights from quantum mechanical and combined quantum mechanical/molecular mechanical simulations as well as other free energy methods and highlight consensus viewpoints as well as remaining controversies. The second part provides a very brief overview of new insights emerging from large-scale molecular dynamics simulations. We conclude with a perspective regarding future studies of Ras where computational approaches will likely play an active role.

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A mechanism of catalyzed GTP hydrolysis by Ras protein through magnesium ion

Cited by 9 publications

References 34 publications

Why nature really chose phosphate

Why nature really chose phosphate

Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

Overview of simulation studies on the enzymatic activity and conformational dynamics of the GTPase Ras

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