The Role of Gln61 in HRas GTP Hydrolysis: A Quantum Mechanics/Molecular Mechanics Study

Martín-García, Fernando; Mendieta‐Moreno, Jesús I.; López-Viñas, Eduardo; Gómez‐Puertas, Paulino; Mendieta, Jesús

doi:10.1016/j.bpj.2011.11.4005

Cited by 49 publications

(67 citation statements)

References 24 publications

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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: 85%

“…Later studies used a combined QM/MM (13-15, 17, 18, 21, 22) approach with different sizes of the QM and the MM region, respectively. There is debate regarding whether the pathway is dissociative or associative (2,17,18,22). Furthermore, some calculated activation energies deviate largely from experimental values.…”

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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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confidence: 85%

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confidence: 99%

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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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“…After this, we investigated mechanisms involving a second water molecule in the active site as these have been reported by Martín-García et al (2012) to be pertinent for glutamineassisted GTP hydrolysis in HRAS. To do this, we placed a FIGURE 3.…”

Section: Mutant Proteinmentioning

confidence: 99%

Mechanism of activation of elongation factor Tu by ribosome: Catalytic histidine activates GTP by protonation

Aleksandrov¹,

Field²

2013

RNA

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Elongation factor Tu (EF-Tu) is central to prokaryotic protein synthesis as it has the role of delivering amino-acylated tRNAs to the ribosome. Release of EF-Tu, after correct binding of the EF-Tu:aa-tRNA complex to the ribosome, is initiated by GTP hydrolysis. This reaction, whose mechanism is uncertain, is catalyzed by EF-Tu, but requires activation by the ribosome. There have been a number of mechanistic proposals, including those spurred by a recent X-ray crystallographic analysis of a ribosome:EF-Tu:aatRNA:GTP-analog complex. In this work, we have investigated these and alternative hypotheses, using high-level quantum chemical/molecular mechanical simulations for the wild-type protein and its His85Gln mutant. For both proteins, we find previously unsuggested mechanisms as being preferred, in which residue 85, either His or Gln, directly assists in the reaction. Analysis shows that the RNA has a minor catalytic effect in the wild-type reaction, but plays a significant role in the mutant by greatly stabilizing the reaction's transition state. Given the similarity between EF-Tu and other members of the translational Gprotein family, it is likely that these mechanisms of ribosome-activated GTP hydrolysis are pertinent to all of these proteins.

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“…10 Computational modeling based on quantum mechanics/molecular mechanics (QM/MM) simulations [13][14][15][16] provides an important tool for understanding the reaction mechanisms in enzymes at atomic level. Although different molecular models lead to partly overlapping conclusions when considering the GTP hydrolysis reaction [17][18][19][20][21][22][23][24][25][26][27][28] (see also the recent review articles [29][30][31] ), there is an agreement between the results from diverse approaches. 19,[25][26][27][28] According to these calculation results the catalytic water molecule (Wat) in the enzyme-substrate complex is aligned by the hydrogen bonds with the side chains of two Ras residues, Thr35 and Gln61.…”

Section: Introductionmentioning

confidence: 99%

Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme

et al. 2015

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Molecular mechanisms of hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by the Ras·GAP protein complex are fully investigated by using modern modeling tools. The previously hypothesized stages of the cleavage of the phosphorus-oxygen bond in GTP and the formation of the imide form of catalytic Gln61 from Ras upon creation of Pi are confirmed by using the higher-level quantum-based calculations. The steps of the enzyme regeneration are modeled for the first time, providing a comprehensive description of the catalytic cycle. It is found that for the reaction Ras·GAP·GTP·H 2 O → Ras·GAP·GDP·Pi the highest barriers correspond to the process of regeneration of the active site, but not to the process of substrate cleavage. The specific shape of the energy profile is responsible for an interesting kinetic mechanism of the GTP hydrolysis. The analysis of the process using the first-passage approach and consideration of kinetic equations suggest that the overall reactions rate is a result of the balance between relatively fast transitions and low probability of states from which these transitions are taking place. Our theoretical predictions are in excellent agreement with available experimental observations on GTP hydrolysis rates.3

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The Role of Gln61 in HRas GTP Hydrolysis: A Quantum Mechanics/Molecular Mechanics Study

Cited by 49 publications

References 24 publications

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

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

Mechanism of activation of elongation factor Tu by ribosome: Catalytic histidine activates GTP by protonation

Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme

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