Quantum chemical modeling of the GTP hydrolysis by the RAS–GAP protein complex

We present results of the modeling for the hydrolysis reaction of guanosine triphosphate (GTP) in the RAS–GAP protein complex using essentially ab initio quantum chemistry methods. One of the approaches considers a supermolecular cluster composed of 150 atoms at a consistent quantum level. Another is a hybrid QM/MM method based on the effective fragment potential technique, which describes interactions between quantum and molecular mechanical subsystems at the ab initio level of the theory. Our results show that the GTP hydrolysis in the RAS–GAP protein complex can be modeled by a substrate-assisted catalytic mechanism. We can locate a configuration on the top of the barrier corresponding to the transition state of the hydrolysis reaction such that the straightforward descents from this point lead either to reactants GTP+H₂O or to products guanosine diphosphate (GDP)+H₂PO₄^?. However, in all calculations such a single-step process is characterized by an activation barrier that is too high. Another possibility is a two-step reaction consistent with formation of an intermediate. Here the Pγ-O(Pβ) bond is already broken, but the lytic water molecule is still in the pre-reactive state. We present arguments favoring the assumption that the first step of the GTP hydrolysis reaction in the RAS–GAP protein complex may be assigned to the breaking of the Pγ-O(Pβ) bond prior to the creation of the inorganic phosphate.     

Mechanisms of guanosine triphosphate hydrolysis by Ras and Ras-GAP proteins as rationalized by ab initio QM/MM simulations

The hydrolysis reaction of guanosine triphosphate (GTP) by p21(ras) (Ras) has been modeled by using the ab initio type quantum mechanical-molecular mechanical simulations. Initial geometry configurations have been prompted by atomic coordinates of the crystal structure (PDBID: 1QRA) corresponding to the prehydrolysis state of Ras in complex with GTP. Multiple searches of minimum energy geometry configurations consistent with the hydrogen bond networks have been performed, resulting in a series of stationary points on the potential energy surface for reaction intermediates and transition states. It is shown that the minimum energy reaction path is consistent with an assumption of a two-step mechanism of GTP hydrolysis. At the first stage, a unified action of the nearest residues of Ras and the nearest water molecules results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low barrier (16.7 kcal/mol) transition state TS1. At the second stage, the inorganic phosphate is formed in consequence of proton transfers mediated by two water molecules and assisted by the Gln61 residue from Ras. The highest transition state at this segment, TS3, is estimated to have an energy 7.5 kcal/mol above the enzyme-substrate complex. The results of simulations are compared to the previous findings for the GTP hydrolysis in the Ras-GAP (p21(ras)-p120(GAP)) protein complex. Conclusions of the modeling lead to a better understanding of the anticatalytic effect of cancer causing mutation of Gln61 from Ras, which has been debated in recent years.     

QM/MM modeling the Ras-GAP catalyzed hydrolysis of guanosine triphosphate

The mechanism of the hydrolysis reaction of guanosine triphosphate (GTP) by the protein complex Ras-GAP (p21(ras) - p120(GAP)) has been modeled by the quantum mechanical-molecular mechanical (QM/MM) and ab initio quantum calculations. Initial geometry configurations have been prompted by atomic coordinates of a structural analog (PDBID:1WQ1). It is shown that the minimum energy reaction path is consistent with an assumption of two-step chemical transformations. At the first stage, a unified motion of Arg789 of GAP, Gln61, Thr35 of Ras, and the lytic water molecule results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low-barrier transition state TS1. At the second stage, Gln61 abstracts and releases protons within the subsystem including Gln61, the lytic water molecule and the gamma-phosphate group of GTP through the corresponding transition state TS2. Direct quantum calculations show that, in this particular environment, the reaction GTP + H(2)O --> GDP + H(2)PO(4) (-) can proceed with reasonable activation barriers of less than 15 kcal/mol at every stage. This conclusion leads to a better understanding of the anticatalytic effect of cancer-causing mutations of Ras, which has been debated in recent years.     

Ab initio study of the role of lysine 16 for the molecular switching mechanism of Ras protein p21

Quantum chemical computations using the ab initio molecular orbital (MO) method have been performed to investigate the molecular switching mechanism of Ras protein p21, which has an important role in intracellular signal cascades. Lys(16) was demonstrated to be crucial to the function of Ras p21, and the hydrolysis of GTP to GDP was found to be an one-step reaction. The potential energy barrier of this hydrolysis reaction from GTP to (GDP + P) was calculated to be approximately 42 kcal/mol. The role of GAP (GTPase-activating protein) was also discussed in terms of the delivery of the water molecules required for the hydrolysis.     

Mechanism of the chemical step for the guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor Tu

Elongation factor Tu (EF-Tu), the protein responsible for delivering aminoacyl-tRNAs (aa-tRNAs) to ribosomal A site during translation, belongs to the group of guanosine-nucleotide (GTP/GDP) binding proteins. Its active 'on'-state corresponds to the GTP-bound form, while the inactive 'off'-state corresponds to the GDP-bound form. In this work we focus on the chemical step, GTP+H(2)O-->GDP+Pi, of the hydrolysis mechanism. We apply molecular modeling tools including molecular dynamics simulations and the combined quantum mechanical-molecular mechanical calculations for estimates of reaction energy profiles for two possible arrangements of switch II regions of EF-Tu. In the first case we presumably mimic binding of the ternary complex EF-Tu.GTP.aa-tRNA to the ribosome and allow the histidine (His85) side chain of the protein to approach the reaction active site. In the second case, corresponding to the GTP hydrolysis by EF-Tu alone, the side chain of His85 stays away from the active site, and the chemical reaction GTP+H(2)O-->GDP+Pi proceeds without participation of the histidine but through water molecules. In agreement with the experimental observations which distinguish rate constants for the fast chemical reaction in EF-Tu.GTP.aa-tRNA.ribosome and the slow spontaneous GTP hydrolysis in EF-Tu, we show that the activation energy barrier for the first scenario is considerably lower compared to that of the second case.     

Metastability of microtubules induced by competing internal forces

Recent modeling efforts to estimate energies of tubulin-tubulin bonds shed light on a delicate balance between competing mechanical forces maintaining microtubule walls. Here we formulate two important refinements to the explanation of bond energetics. First, energy surface calculations in the elastic filament approximation reveal a finite stabilizing barrier assumed a simple Lennard-Jones-like potential for protein bonds. The presence of a guanosine triphosphate (GTP) cap represented by straight segments is necessary, as it is predicted for a long time. In the lack of such a cap, the protofilaments are either in an absolutely stable or absolutely unstable state. Second, our calculations show that this barrier appears only if the mechanical energy associated with the conformational change after GTP hydrolysis (curling energy) is larger than the strength of lateral bonds. The overall energy balance we propose supports continuous assembly of GTP dimers, a metastable state in the presence of a finite GTP cap and energetically driven disassembly of guanosine diphosphate protofilaments.     

Is there a rate-limiting step before GTP cleavage by H-ras p21?

A slow fluorescence change of the complex between ras p21 and the fluorescent GTP analogue 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-triphosphate (mGTP) has been postulated to be a signal arising from a step which is rate limiting and precedes the actual GTP hydrolysis reaction [Neal, S. E., Eccleston, J. F., & Webb, M. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565]. We have now shown that the rate of the fluorescence change is accelerated by GTPase-activating protein (GAP) in the same manner as that of the GTP cleavage reaction. In contrast, a faster fluorescence change of smaller amplitude seen in the complex between p21 and the uncleavable 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-O-(beta,gamma-imidotriphosphate) (mGppNHp) is not affected by GAP. The corresponding fluorescent derivative of guanosine 5'-O-(gamma-thiotriphosphate) (mGTP gamma S) shows a very slow fluorescence change after binding to p21, and this rate is also accelerated significantly by GAP. Hydrolysis of GTP gamma S is similarly slow, and it is accelerated by GAP in a similar manner to the fluorescence change. The results are interpreted to indicate that the fluorescence change occurs either at the hydrolysis step or on release of inorganic phosphate or thiophosphate but does not occur in a rate-limiting step preceding hydrolysis.     

Computational characterization of the chemical step in the GTP hydrolysis by Ras‐GAP for the wild‐type and G13V mutated Ras

The free energy profiles for the chemical reaction of the guanosine triphosphate hydrolysis GTP + H₂O → GDP + Pi by Ras‐GAP for the wild‐type and G13V mutated Ras were computed by using molecular dynamics protocols with the QM(ab initio)/MM potentials. The results are consistent with the recent measurements of reaction kinetics in Ras‐GAP showing about two‐order reduction of the rate constant upon G13V mutation in Ras: the computed activation barrier on the free energy profile is increased by 3 kcal/mol upon the G13V replacement. The major reason for a higher energy barrier is a shift of the “arginine finger” (R789 from GAP) from the favorable position in the active site. The results of simulations provide support for the mechanism of the reference reaction according to which the Q61 side chain directly participates in chemical transformations at the proton transfer stage. Proteins 2015; 83:1046–1053. © 2015 Wiley Periodicals, Inc.     

Acetylcholinesterase: mechanisms of covalent inhibition of H447I mutant determined by computational analyses

The reaction mechanisms of two inhibitor TFK(+) and TFK(0) binding to H447I mutant mouse acetylcholinesterase (mAChE) have been investigated by using a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach and classical molecular dynamics (MD) simulations. TFK(+) binding to the H447I mutant may proceed with a different reaction mechanism from the wild-type. A water molecule takes over the role of His447 and participates in the bond breaking and forming as a "charge relayer". Unlike in the wild-type mAChE case, Glu334, a conserved residue from the catalytic triad, acts as a catalytic base in the reaction. The calculated energy barrier for this reaction is about 8kcal/mol. These predictions await experimental verification. In the case of the neutral ligand TFK(0), however, multiple MD simulations on the TFK(0)/H447I complex reveal that none of the water molecules can be retained in the active site as a "catalytic" water. Taken together our computational studies confirm that TFK(0) is almost inactive in the H447I mutant, and also provide detailed mechanistic insights into the experimental observations.     

The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis

The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2-GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.     

Guanine nucleotides stimulate soluble phosphoinositide-specific phospholipase C in the absence of membranes

The effect of guanine nucleotides on platelet and calf brain cytosolic phospholipase C was examined in the absence of membranes or detergents in an assay using labeled lipid vesicles. Guanine nucleotides stimulate hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate [( 3H]PtdIns-4,5-P2) catalyzed both by enzyme from human platelets and by partially purified enzyme from calf brain. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was the most potent guanine nucleotide with a half-maximal stimulation at 1-10 microM, followed by guanosine 5'-(beta, gamma-imido)triphosphate greater than GTP greater than GDP = guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(2-thiodiphosphate) was able to reverse the GTP gamma S-mediated stimulation. NaF also stimulated phospholipase C activity, further implying a role for a guanine nucleotide-binding protein. In the presence of GTP gamma S, the enzyme cleaved PtdIns-4,5-P2 at higher pH values, and the need for calcium ions was reduced 100-fold. The stimulation of PtdIns-4,5-P2 hydrolysis by GTP gamma S ranged from 2 to 25-fold under various conditions, whereas hydrolysis of [3H]phosphatidylinositol was only slightly affected by guanine nucleotides. We propose that a soluble guanine nucleotide-dependent protein activates phospholipase C to hydrolyze its initial substrate in the sequence of phosphoinositide-derived messenger generation.     

Steady state kinetic analysis of the mechanism of guanosine triphosphate hydrolysis catalyzed by Escherichia coli elongation factor G and the ribosome.

The mechanism of guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor G and the ribosome in the absence of other participants in protein synthesis was examined by steady-state kinetic analysis. Optimal hydrolytic conditions were determined to be approximately pH 8.0, 20 mM Mg2+, and 80 mM NH4+. Kinetic analyses were performed under these conditions at constant elongation factor G concentrations and variable ribosome and GTP concentrations. The resulting double-reciprocal plots in conjunction with the inhibition patterns obtained with GDP indicated that the reaction occurs by an ordered mechanism in which GTP is the leading obligatory substrate. Dissociation constants for GTP and guanosine diphosphate (GDP), as well as limiting Michaelis constants for GTP and ribosomes, were calculated from the double-reciprocal plots. These values are: KSGTP = 37.0 muM, KSGDP = 16.5 muKMGTP = 8.0 muM, KMR = 0.22 muM. Inhibition was also observed at high ribosomal concentrations and suggests that inhibition was due both to the decreased breakdown of the tertiary elongation factor G-GDP-ribosome posthydrolytic complex and to the formation of a nonproductive elongation factor G-ribosome complex. A sequential mechanism with a dead-end elongation factor G-ribosome complex has been constructed to describe the hydrolysis of GTP catalyzed by elongation factor G and the ribosome.     

Conformational changes in the GTPase modules of the signal reception particle and its receptor drive initiation of protein translocation

During cotranslational protein targeting, two guanosine triphosphatase (GTPase) in the signal recognition particle (SRP) and its receptor (SR) form a unique complex in which hydrolyses of both guanosine triphosphates (GTP) are activated in a shared active site. It was thought that GTP hydrolysis drives the recycling of SRP and SR, but is not crucial for protein targeting. Here, we examined the translocation efficiency of mutant GTPases that block the interaction between SRP and SR at specific stages. Surprisingly, mutants that allow SRP-SR complex assembly but block GTPase activation severely compromise protein translocation. These mutations map to the highly conserved insertion box domain loops that rearrange upon complex formation to form multiple catalytic interactions with the two GTPs. Thus, although GTP hydrolysis is not required, the molecular rearrangements that lead to GTPase activation are essential for protein targeting. Most importantly, our results show that an elaborate rearrangement within the SRP-SR GTPase complex is required to drive the unloading and initiate translocation of cargo proteins.     

The role of magnesium for geometry and charge in GTP hydrolysis, revealed by quantum mechanics/molecular mechanics simulations

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and P(i) in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and P(i) release back to the diphosphate. Furthermore, the Ras·Mg(2+) complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger.     

Coherent singlet-triplet transitions at the initial step of tubulin assembly into microtubules

Quantum chemistry calculations [DFT-B3LYP QM/MM method, 6-31G** basis set, + ab initio molecular dynamics] were used to study the action of Mg2+ on tubulin properties. It was shown that the hydration of the guanosine triphosphate-tubulin forms a protein zone structure, which includes a electron-occupied zone and a conductivity zone. The binding of Mg2+ to guanosine triphosphate-tubulin results in the unpairing of electrons in the occupied zone (triplet state formation) followed by their transition to the conductivity zone in which the inversion of spin occurs (singlet state formation). The formation of triplet state is the initial step in the subsequent protein dynamics in the picosecond range of time. The dynamics shows up as a coherent oscillating transition of tubulin between the triplet and singlet states, which is evidence of a simultaneous adjustment between nuclear and electron configurations of the protein (ab initio molecular dynamics calculations). The barrier between the triplet and singlet states does not exceed 0.60 kcal x mol(-1). The barrier overcome is considered as electron tunneling through the Fermi surface, which separates the occupied and conductivity zones. Zone formation occurs in the presence of the shell of biological water surrounding the protein.     

GTP degradation to guanine catalyzed by ribosomal subunits and microsomal-wash factors.

Ribosomes from stringent strains of bacteria generate (p)ppGpp if incubated with uncharged tRNA, a ribosomal wash fraction, GTP and ATP. By contrast, an analogous system from rat liver does not transform GTP to (p)ppGpp but degrades it to guanine. The reaction requires the ribosomal subunits, a 40 000-Mr and a 60 000-Mr microsomal wash protein factor and is inhibited if the ribosomal A-site is charged with aminoacyl tRNA. The degradation of GTP to guanine occurs in the following four distinct reaction steps: (a) hydrolysis of GTP to GDP plus Pi, (b) hydrolysis of GDP to GMP plus Pi, (c) hydrolysis of GMP to guanosine plus Pi, (d) hydrolysis of guanosine to guanine plus ribose. The reaction step (a) is inhibited by fusidic acid, cycloheximide, emetine, tetracycline and puromycin. The hydrolysis of GDP is inhibited strongly by fusidic acid, emetine and tetracycline. A putative physiological significance of this ribosome-dependent pathway in the processes of growth control of animal cells under conditions of amino acid deprivation is discussed.     

Simulated <Superscript>18</Superscript>O kinetic isotope effects in enzymatic hydrolysis of guanosine triphosphate

We compare the computed on the base of quantum mechanical-molecular mechanical (QM/MM) modeling kinetic isotope effects (KIEs) for a series of the ¹⁸O-labeled substrates in enzymatic hydrolysis of guanosine triphosphate (GTP) with those measured experimentally. Following the quantitative structure-activity relationship concept, we introduce the correlation between KIEs and structure of substrates with the help of a labeling index, which also aids better imaging of presentation of both experimental and theoretical data. An evident correlation of the computed and measured KIEs provides support to the predominantly dissociative-type reaction mechanism of enzymatic GTP hydrolysis predicted in QM/MM simulations.     

Guanosine 5'-O-(3-thiotriphosphate) as an analog of GTP in protein biosynthesis. The effects of temperature and polycations on the accuracy of initial recognition of aminoacyl-tRNA ternary complexes by ribosomes

Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) is a good analog of GTP in the reactions leading to the formation of a peptide bond in protein biosynthesis. It forms binary and ternary complexes with elongation factor Tu (EF-Tu), and with EF-Tu and aminoacyl-tRNA (aa-tRNA). In addition, it stimulates aa-tRNA binding to ribosomes. Although GTP gamma S hydrolysis is more than three orders of magnitude slower than GTP hydrolysis, both reactions are dependent on the formation of a noncovalent complex (RS X TC) between mRNA-programmed ribosomes and ternary complex, and the complexes resulting from that hydrolysis are intermediates in peptide formation. The rate of dissociation of the ribosome X EF-Tu X GTP gamma S X aa-tRNA complex was determined from the rate of labeled peptide formation in the presence of an unlabeled ternary complex chase. This rate (2.2 X 10(-3) s-1) is similar to that determined previously (Thompson, R.C., and Karim, A.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4922-4926) from the progress of GTP gamma S hydrolysis. The effects of temperature and polycation concentration on this rate constant and that for GTP gamma S hydrolysis are reported. The rate constants measured are consistent with a kinetic rather than thermodynamic limit on the accuracy of the aa-tRNA selection in vivo.     

The roles of phospholipase D and a GTP-binding protein in guanosine 5''-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5'-[beta gamma-imido]triphosphate and guanosine 5'-[alpha beta-methylene]triphosphate, but not adenosine 5'-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5'-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.