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Clarifying the Catalytic Mechanism of Human Glutamine Synthetase: A QM/MM Study Cátia Moreira, Maria João Ramos, and Pedro A. Fernandes J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 7, 2017

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The Journal of Physical Chemistry

Clarifying the Catalytic Mechanism of Human Glutamine Synthetase: A QM/MM Study Cátia Moreira, Maria J. Ramos and Pedro A. Fernandes* UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, s/n, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto (Portugal). E-mail: [email protected]

ACS Paragon Plus Environment

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Abstract:

Glutamine synthetase (GS) is a crucial enzyme responsible for the elimination of both neurotoxic glutamate and toxic ammonium, by combining them into glutamine. Alterations on the GS activity are associated with severe liver and neurodegenerative diseases and its absence or malformation results in death. In this work, the catalytic mechanism of human GS has been investigated with high-level QM/MM calculations, showing a two-phase reaction cycle. During phase 1, GS activates the reactants (NH4+ and glutamate) with extreme efficiency, through NH4+ deprotonation by E305 and glutamate phosphorylation by ATP, in two spontaneous and barrierless reactions. At phase 2, NH3 attacks the γ-glutamyl phosphate being concomitantly deprotonated by the leaving PO43-, forming the glutamine and HPO42- products. The second phase contains the rate limiting step, with a ∆G‡ of 19.2 kcal·mol-1 associated to the nucleophilic substitution of the phosphate by NH3. The final reaction free energy is -34.5 kcal·mol-1. Both phases are exergonic, the first by -22.9 kcal·mol-1 and the second by -11.6 kcal·mol-1. Direct NH4+ attack is shown to be inefficient; the possible bases that perform the NH4+ deprotonation were systematically investigated. Negative E305 was shown to be the only one possibly responsible for NH4+ deprotonation. Altogether these results provide a clear atomic level picture of the reaction cycle of GS, consistent with experimental and theoretical studies on GS of this and other organisms, and provide the necessary insights for the development of more specific therapeutic GS inhibitors.


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Introduction Glutamine synthetase (GS, EC 6.3.1.2, also known as γ-glutamyl:ammonia ligase) is a metalloenzyme that combines ammonium and glutamate into glutamine, at the expense of ATP 1. This reaction plays a key role on the inorganic nitrogen incorporation into organic compounds, with the end product (glutamine) serving as a building block for the biosynthesis of several metabolites2. The importance of this enzyme to life itself is undeniable, being found in all living organisms. Depending on the organism or cellular location, GS can vary in sequence and structure, being categorized under 3 types - GSI, GSII and GSIII - that can be further subdivided into several subtypes and sub-subtypes (for instance, plant GSII can be divided into cytosolic GS1 or plastidic GS2, and GS1 can be further subdivided into GS1α, GS1β and GS1γ)3-5. In mammals, GSII is the most common type of GS found, characterized by an amino acid sequence of around 350 residues1. This type of GS forms a homodecamer structure organized in two pentameric rings, connected by an interacting loop that delimits the C- and N-terminus of each monomer (Figure S1)6. The active site is inserted in the interface between adjacent monomers, within the same pentameric ring, forming a channel that resembles a bifunnel, with its narrower center part forming the “divalent metal binding region” where three divalent metals usually Mg2+ or Mn2+, are found coordinated to conserved regions of GS1.The two larger parts of the bifunnel form both the ATP cofactor and the glutamate substrate binding regions (also called the nucleotide and the aminoacid binding pockets; Figure 1). The nucleotide binding pocket is bigger and less conserved than the aminoacids binding pocket.


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Figure 1. I) Active site location in between two adjacent monomers (blue and green transparent surfaces), with C-domain represented in green cartoon and N-domain represented in blue cartoon; and II) QM model of human GS; for the sake of clarity only ammonium hydrogens were included. The Mg2+ ions (MgA2+ MgB2+ MgC2+) are indicated by the pink spheres labelled A-C and the waters are identified by red spheres labelled 1 and 2. The substrates and residues actively participating in the reaction mechanism are highlighted in ball and stick representation.


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The GS function may be different among distinct organisms or between distinct locations of the same organism. Human GS (hsGS) activity has been detected on skin, peripheral lymphocytes, liver, brain and digestive tract (mostly stomach) 2. In most tissues, the main role of GS is the elimination of toxic ammonia, in particular in the liver, where its malfunction is associated with liver cirrhosis, chronic hepatitis B and C, some types of hyperplasia and some neoplasms

7, 8

. In the brain, hsGS monitors neurotoxic glutamate levels by converting glutamate

into less toxic glutamine. Malfunction of brain hsGS is associated with several neurological diseases such as Alzheimer’s, epilepsy, seizures, glioblastoma multiform, anxiety and depression 9-16

. Glaucoma is another disease associated with this enzyme, this time in the eye 17. Total lack

of GS or its malformation leads to neonatal death 2, 18. Huge efforts are being made to find new GS inhibitors with both clinical and agricultural applications. One plant GS inhibitor (glufosinate) is one of the most widely used herbicides across the planet

19

. Several studies proved that bacterial GS is a promising target for the

development of drugs against tuberculosis 20, 21, with already several inhibitors being designed 2228

. The GS inhibitors obtained so far target the glutamate or the ATP binding regions, with some

of them being already co-crystallized within the GS enzymes

22, 25, 29, 30

. Phosphinotricin and L-

Met-S-sulfoximine are the most used inhibitors targeting the aminoacids binding site, acting as transition state (TS) analogue suicide inhibitors. However effective, these inhibitors lack selectively for specific organisms. As GS is fundamental for all living organisms, its unselective inhibition can be catastrophic. Recent advances in Mycobacterium tuberculosis GS (mtGS) inhibition had led to the design of more selective inhibitors, targeting a less conserved ATP binding site, presenting themselves as more promising therapeutic agents 20-23, 31.


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In this scenario, a more detailed view about the way GS works could give the final hint to unlock the discovery of new and more selective and safe inhibitors, to use either as herbicides or antibiotics. There are still a lot of questions to be asked regarding the catalytic and inhibition mechanisms of GS that need urgent answers. Many studies point out to the fact that different GSs can behave differently, such as being more or less susceptible to a given inhibitor 32, but the reason for this is not clear. The mtGS architecture of the ATP binding site is quite different from human GS (hsGS), and thus it may, or may not, influence the mechanism of the reaction. Additionally, some studies point towards the existence of a tetrahedral intermediate during the condensation step of the catalytic cycle 33, with ammonia and phosphate bound to glutamate, that was never found. The current understanding of the action mechanism of GSs has been gathered from crystallographic structures

34, 35

, kinetic studies

36-39

and, more recently, two computational

studies: 1) our previous study on the bacterial type I mtGS by QM/MM 40; and 2) the very recent hsGS type II studied by MD-QM/MM

41

. Here, to study the hsGS enzyme, we apply a similar

methodology to the one that we have used on the mtGS reaction mechanism, and compare the results to those obtained previously in this and other organisms.

Theoretical Methods The model used throughout the calculations was built from the human GS crystal structure (hsGS; PDB accession code 2QC8, 2.6 Å resolution 1). This structure contains L-methionine-Ssulfoximine phosphate (a transition state like inhibitor), ADP and three Mn2+. Since the active site is located in between two adjacent subunits, a dimer was selected to serve as model (Figure 1). Mn2+ ions were replaced with Mg2+ ions since it is described that Mg2+ ions are the ones


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present on bovine brain

42

and enhance the hsGS activity

43

. L-methionine-S-sulfoximine

phosphate served as a base to model the first intermediate state, γ-glutamyl phosphate and ammonium, by direct substitution of the of δ-sulphur, ε-nitrogen and ε-carbon atoms of the inhibitor by carbon, oxygen and nitrogen, on Avogadro 44 software. The bond between δ-sulphur and ε-nitrogen in the L-methionine-S-sulfoximine phosphate was deleted to start with free ammonium. The protonation states suitable for hsGS were first investigated by PROPKA

45, 46

in order to

point out residues that do not follow the standard protonation state at pH 7. According to this software, only residue E305 should have an atypical protonation state (i.e. protonated). However, after carefully checking of the E305 environment we conclude that the standard protonation state represents the most probable protonation. Ligands and metals (here Mg2+ and NH4+) can influence greatly the protonation state of protein residues, and the program does not take them into account. As E305 is H-bonded to NH4+ we kept it negative. Either way, a second model was created with the sole difference of having E305 protonated. All hydrogens, Na+ counter ions and TIP3P water molecules were added using the Leap module of the AMBER 12 package 47 in order to protonate, neutralize the charge and solvate the system for further minimizations. The protein residues were described by the ff99SB force field

48, 49

and the ligands (ADP, γ-glutamyl

phosphate and ammonium) were parameterized as stated in our previous study

40

. To relax the

geometry of the model, and eradicate eventual clashes, energy minimizations were performed, using the SANDER module of the AMBER 12 package with the same 4 stages protocol described in 40 that gradually releases the restrains of the model. The final relaxed and minimized models were used to build the QM/MM model used on this study. Note that from the minimized model, we subtracted both the Na+ counter ions and most of


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the water molecules, keeping only the protein, metal ions, the catalytic ligands and some important waters that were located inside the active site. The QM/MM model built on gaussview software 50 contains two layers following the ONIOM partition scheme 51-53. The active site main residues and ligands were included in the QM layer. Notice that in most cases, only the atoms that were needed to perform the reactions or to correctly describe electronic rearrangements were selected for this layer; the remaining portion of the residue/ligand placed in the MM layer had a hydrogen link atom saturating the QM atoms with incomplete valences. The QM layer selected atoms include side chains of D63, E134, E136, E196, E203, H253, E305 (both negative and neutral protonation state), R340 and E338 plus three Mg2+, two water molecules, ammonium and the reactive portion of γ-glutamyl phosphate and ADP (a complete list of the atoms included in the QM layer can be found in SI, and a representation of the atoms included in the QM part can be consulted on Figure 1). The remaining atoms were included in the MM layer and the ones at a distance larger than 15Å from the QM layer were frozen (the selection was made in Pymol

54

).

The list of frozen atoms can also be found in SI. The final two layer models (Figure 1) contains 101/102 atoms in the QM layer from a total of 11129/11131 atoms in the complete model (the difference in the number of atoms is due to the protonation state of E305), with the interactions between the layers calculated by the electrostatic embedding scheme. All QM/MM calculations were made within Gaussian 09 55. The ff99SB force field described the MM layer. The B3LYP functional

56, 57

together with 6-31G (d) basis set

58

was used in the

QM layer on geometry optimizations, and to calculate zero point energies, vibrational frequencies and thermal and entropic contributions to the free energy. The exploration of the reaction started at the fully optimized structure of Int1 (defined by the presence of γ-glutamyl phosphate, ADP and ammonia). From Int1, the reaction could take two direction: forwards, to


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form the glutamine by ammonia attack on the γ-glutamyl phosphate; backwards, to form the reactants (ATP, glutamate and ammonium ion). Adiabatic mappings along the putative reaction coordinates were performed to attain good guesses for the remaining stationary states of this reaction, with intermediates and transition states later fully optimized and confirmed by frequency calculations. These calculations also allow attaining the Gibbs energy at the physiological temperature by calculation of the zero-point energy, the entropy and the thermal energy (all at 310.15K) contributions, within the particle in a box/rigid rotor/harmonic oscillator formalism (with great applicability in calculations of entropy and free energy of a system with the single X-ray conformation). When all the relevant intermediates and transition states were found and optimized, the M06 density functional

59

with the D3 dispersion correction

60

and basis set 6-311++G (2d, 2p) were

employed to attain the final energy profile of this reaction, at the M06-D3/6-311++G (2d, 2p):ff99SB//B3LYP/6-31G(d):ff99SB level. Hirschfield

61, 62

atomic charges for the QM portion

of the model were calculated to infer the charge variation along the reaction. Note that the typical energy errors inherent to this methodology are around 2-5 kcal.mol-1 63.

Results and discussion Phase 1: from the reactants to phosphorylated glutamate (Int1) and ammonia

Glutamate, ammonium and ATP are the reactants needed by GS for phase 1 of the reaction. Kinetic studies have shown that the absence of ammonium only impairs the formation of glutamine, not the phosphorylation of glutamate

36

. As such, the activation of ammonium

(through deprotonation) and of glutamate (through phosphorylation) are independent reactions.


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Crystallographic experiments always failed to catch ATP and glutamate (or a substrate analogue) at the active site, always finding ADP and a phosphate or ADP and a phosphorylated substrate analogue instead 1, 35, 64. Our studies provided an explanation for this, and revealed that hsGS is exceedingly efficient in activating its substrates. Both ammonia and ammonium can enter the active site. Ammonia is more reactive as it has a free pair of electrons to perform a nucleophilic attack to the glutamate’s Cγ. On the other hand, ammonium is the most abundant species in physiological conditions (pKa of 9.3; NH4+:NH3 ratio around 63:1 at physiological pH of 7.5) and interacts more strongly with the very negative active site pocket formed by D63, E196 and E305. So, as pointed out in previous studies on mtGS

40

,

ammonium will enter the hsGS active site and, when inside, it should be deprotonated by one of the basic residues around it, namely E305, D63, E196. When we modeled the system we saw that once ammonium enters the active site it is spontaneously deprotonated by the negative E305, in a barrierless reaction. We tried to move the proton from ammonium to all the bases present at the active site, but no system with other protonated base was a stationary point. In all cases, except E305, the proton returned to ammonium upon geometry optimization. The formation of a hydrogen bond between E305 and D63 was observed subsequently, and seemed to be favored in the presence of ADP and γ-glutamyl phosphate (int1), probably due to the proximity of the phosphate group, that was better positioned to stabilize ammonia (the distance of interaction between phosphate oxygen and ammonia decreased from 4.22 Å to 3.36 Å with the rotation of E305 hydrogen towards interaction with D63). This rotation of E305 implies a loss of hydrogen bond between ammonia and E305, inducing a rearrangement of the ammonia


10

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that directs the ammonia free lone pair of electrons to perform the attack to the substrate. Ammonia also becomes closer to carbon Cγ, which will suffer a nucleophilic substitution. These results are in good agreement with the recent catalytic study on hsGS

41

, where it was

observed that ammonium loses a proton to negative E305, but differs from the results of the reaction mechanism of mtGS

40

, where D54 (equivalent to hsGS D63) was responsible for the

deprotonation of ammonium. However, in the study of mtGS reaction mechanism, the protonated D54 keeps hydrogen bonded to ammonia throughout the whole nucleophilic attack. It has been speculated that E305 could be protonated at the beginning of the cycle. We tried to repeat the calculations starting with a protonated E305 but in this case no residue was capable to deprotonate ammonium. Therefore, the latter protonation state seems to be very inefficient for catalysis. Next, we investigated the phosphate transfer from ATP to the glutamate’s Oδ. We did the study in the reverse direction, by transferring back to ADP the phosphate group of γ-glutamyl phosphate, to originate the reactants’ glutamate and ATP. This was more convenient as we could start the calculations from a crystal structure of this intermediate. In our previous study of mtGS, where a similar methodology was applied, this reaction was found to be barrierless, once the complex was formed in the active site; neither a stable reactant (React) nor a transition state (TS1) was found on the potential energy surface 40. However, very recent studies on the human enzyme found both the React and TS1, with a reaction free energy of -8 kcal·mol-1 and a free energy barrier of 5 kcal·mol-1, at the SCC-DFTB/MM level of theory using multiple steered molecular dynamics (MSMD) calculations

41

. Here we were also able to

fully optimize both the React and TS1 at the M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/631G(d):ff99SB level of theory, but the reaction proceeds in an essentially barrierless fashion


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because the free energy (0.5 kcal·mol-1) barrier is smaller than kBT. The transition state was characterized by an imaginary frequency of 136.8 i·cm-1. TS1 has a bipiramidal trigonal geometry and involves the transient formation of PO32- (Figure 2). From React to TS1 the phosphate bond elongated from 1.86 Å to 2.10 Å (d1 on figure 2) and the distance between the phosphorous and the substrate Oδ (d2 on figure 2) shortened from 3.01 Å to 2.61 Å. At Int1 the bond length between phosphorus and Oδ partner reached 1.74 Å.

Figure 2. I) Schematic representation of phosphate transfer reaction mechanism from ATP to glutamate; II) representation of the TS1 geometry. For the sake of clarity, only the substrates (R1 = glutamate/γ-glutamyl phosphate; R2 = ATP/ADP) and Mg2+ ions were included. Black dash lines illustrate the distances directly involved in bond breaking and bond formation; and III) Values for the distances and charges illustrated on (II) for React, TS1 and Int1.

As ATP is dephosphorylated, structural rearrangements occurred on some of the residues involved in the Mg2+ coordination (figure S2). Besides phosphate transfer, a water molecule


12

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(Water 2, Fig. S2) was transferred from the coordination sphere of MgB2+ to that of MgA2+. This is the first time that a water shift from the coordination shell of MgB2+ to MgA2+ is reported. The importance of Mg2+ in the GS active site is undeniable, playing both structural and catalytic roles 43

, by stabilizing the negative charges of both reactants and assisting this transfer reaction

between two negative groups, that would repel themselves if in solution. The reaction free energy was -22.9 kcal·mol-1 and the free energy barrier was 0.5 kcal·mol-1, i.e. smaller than kBT. This free energy profile clearly explains why the complex between ATP and the substrate has never been captured in crystallographic studies

1, 35, 64

, and is in agreement

with the previous mtGS reaction mechanism study 40.This also shows that GS has the ability to eliminate the activation free energy for the phosphate transfer, pushing the reaction to become only dependent on the binding of the reactants.

Phase 2: from phosphorylated glutamate to glutamine

Once both reactants were activated – glutamate phosphorylated and ammonium deprotonated – the second, limiting phase of the reaction cycle took place. A linear transition scan along the CγNH3 distance was performed, from which a maximum energy point (TS2) was further fully optimized and characterized. The optimized TS2 was located at a Cγ-NH3 distance of 1.57 Å and Cγ-OPO33- distance of 1.83 Å, associated to an antisymmetric vibration (156.9 i·cm-1) of the a Cγ-NH3 and Cγ-OPO33- bonds (Figure 3). NH3 was spontaneously deprotonated by the leaving phosphate upon binding the Cγ. The NH3 proton was still not transferred to the leaving phosphate at TS2 (1.69 Å), keeping an essentially intact bond to NH3 at this stage (1.06 Å), but was fully transferred at the reaction products. This transition state is similar to the one found in


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similar studies on mtGS 40. An activation free energy of 19.2 kcal·mol-1 was determined for TS2, which is in good agreement with both experimental studies (15-17 kcal·mol-1) computational studies (19-21.5 kcal·mol-1)

40, 41

36-39

and

, pointing to this reaction as being the limiting

step of GS catalysis. The reaction free energy is -11.6 kcal·mol-1. The postulated tetrahedral intermediate33 was shown not to exist, not even at TS2, where the phosphate is mostly unbound while ammonia is mostly bound. The double bond between the Cγ and the non-phosphorylated Oδ of the substrate has never been elongated from the typical double bond C=O distance (1.21 Å), even at TS2 (where it takes a value of 1.23 Å). This points to a concerted, slightly dissociative SN2 reaction (plus a coupled, asynchronous, proton transfer), with a quasisynchronous replacement of the two bonds, and always preserving the sp2 hybridization at Cγ. This reaction ends with the formation of the neutral glutamine product (Prod), and a free energy that lies below Int1. This is new in computational studies of GS, in which the reaction free energy of the limiting step was always calculated to be positive 40, 41.

Figure 3. I) schematic representation of glutamate and ammonia condensation into glutamine; II) representation of TS2 geometry for the reactive species. Black dashed lines with white arrows denominated dA-dB illustrate the distances directly involved in bond breaking and bond formation of TS2. Black lines and arrows named dC-dD represent important distances related to


14

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proton transfer from glutamine to phosphate; III) Table containing all the important distances illustrated on (II) for Int1, TS2 and Prod.

Charge analysis of the QM system shows slight alterations on several residues located in the aminoacids binding pocket (Table S2). These charge variations can be associated once more with geometry adjustments of the system to the glutamine formation (Figure S3). H253 strengthens its interaction with water 2 by changing the N--H distance from 2.13 Å to 1.72 Å; the E196 interaction with the amide group of glutamine becomes stronger (changing from 3.52 Å on int1 to 2.03 Å on prod); R340 weakens its interaction with glutamine and phosphate; and E203 moves towards the H253 residue. The more prominent changes in atomic charges are observed in the atoms directly involved on the nucleophilic substitution. At TS2, the donation of the ammonia lone pair to Cγ induces an increase in positive charge on ammonia, of +0.31 a.u.; simultaneously the phosphate takes the electron pair that was making the bond to the Oδ, and its negative charge increases by -0.46 a.u.. In the products, as ammonia binds Cγ and is deprotonated by the leaving phosphate, it loses again all its excess of positive charge (-0.04 a.u. in relation to Int1) and the leaving phosphate loses its excess of negative charge, upon protonation (-0.16 a.u. in relation to Int1). Coordinates and charge information for all optimized geometries are available in SI. To be on the safe side we also tested the hypothesis of an initial ammonium attack coupled with a double proton transfer. Therefore, we have started from Int1, obtained in the active site that has E305 protonated from the beginning, as hypothesized by others41; we started from the much less reactive ammonium molecule, as in this active site protonation state, no base is able to deprotonate ammonium. A linear transit scan along the Cγ-NH4+ distance was performed, and an ammonium proton was transferred to a phosphate oxygen concomitantly with ammonia binding


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to the substrate Cγ. The Cγ-bound phosphate was eliminated in a concerted deportonation+SN2 reaction. The cationic glutamine lost its amide proton to E196 on its way to the products (Figure S4). The transition state for this reaction was not further optimized as the high activation energy associated with the potential energy profile (50 kcal.mol-1) shows that this alternative mechanism is not a valid hypothesis for the hsGS catalytic cycle, and cannot compete with the reaction mechanism shown in Figure 4. Similar results were attained on recent computational studies on hsGS reaction mechanism, at SCC-DFTB level of theory 41.

Figure 4. Free energy profile of the phosphate transfer reaction (phase 1) and glutamine formation reaction (phase 2). ∆G (in kcal·mol-1), ligand geometries and the reaction important distances (in Å) are shown for all stationary states of the free energy profile.

Conclusions


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The efforts to uncover the ticks and tricks of GS catalysis are paying off, with the accomplishment of already three independent studies on GS reaction mechanisms at atomic level, one in mtGS and two in the hsGS. Here we consolidate and clarify the information on the reaction mechanism of GS enzymes by the study of the hsGS using a high level DFT/MM methodology coupled to adiabatic mapping. We observe a two-phase mechanism: phase 1) activation of the reactants (ATP, Glutamate and ammonium); and phase 2) glutamine formation. In phase 1 was described the phosphate transfer reaction from the ATP to the glutamate, forming γ-glutamyl phosphate, with a reaction free energy of -22.9 kcal.mol-1 and a negligible activation barrier (0.5 kcal.mol-1) making this reaction spontaneous and barrierless. The deprotonation of ammonium by E305 is also barrierless. Therefore, this enzyme has the amazing property of activating the two reactants, which it will condensate later on, at the diffusion speed limit. In phase 2, ammonia performs a nucleophilic attack on the γ-carbon of the γ-glutamyl phosphate, concerted with phosphate release and glutamine amide deprotonation by the leaving phosphate. The combination of these two transformations, SN2 reaction plus deprotonation, involves an activation free energy of 19.2 kcal.mol-1 and a reaction free energy of -11.6 kcal.mol1

. The activation free energy is in good agreement with both experimental (15-17 kcal.mol-1) 36-39

and computational studies (19 kcal.mol-1 on hsGS and 21.5 kcal.mol-1 on mtGS)

40, 41

on GS

catalysis. Figure 4 shows the overall free energy profile. This reaction mechanism was attained starting with negative E305 and ammonium in the active site. The reaction does not take place starting from protonated E305. In this case ammonium cannot be deprotonated, and the attack of ammonium to the γ-glutamyl phosphate involved a barrier of over 50 kcal.mol-1. This alternative reaction mechanism is very similar to


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the one described in a recent computational study on hsGS at SCC-DFTB level of theory

41

,

where ammonium loses a proton to the leaving phosphate group at the same time that ammonia binds γ-glutamyl phosphate and phosphate is released in a SN2 reaction, followed by glutamine amide

group

deprotonation

by

E196.

However,

at

the

M06-D3/6-311++G

(2d,

2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory such alternative is not viable. Deprotonation of ammonium by the glutamyl phosphate leads to a non-stationary, very highenergy structure, which reverts to the reactants through a barrierless pathway. The same happened with ammonium deprotonation by nearby Asp/Glu residues other than Glu305. The only viable pathway at the M06-D3/6-311++G (2d, 2p):ff99SB//B3LYP/6-31G(d):ff99SB level is the one proposed here. In summary, this study further clarifies the catalytic mechanism of hsGS with high-level DFT/MM methods. The results are in agreement with experimental and theoretical studies, and shall be of great help for the development of selective anti-tuberculosis drugs and safe herbicides.


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ASSOCIATED CONTENT Supporting Information. A PDF document containing all SI figures, tables and schemes and a zipped folder containing PDB files with coordinates and charges are available free of charge.

AUTHOR INFORMATION Author Contributions The manuscript was written by C. Moreira and revised through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work received financial support from the following institutions: European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/MULTI/04378/2013. C.M. thanks the FCT for her SFRH/BD/84016/2012 grant.


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References 1. Krajewski, W.; Collins, R.; Holmberg-Schiavone, L.; Jones, T. A.; Karlberg, T.; Mowbray, S. L., Crystal Structures of Mammalian Glutamine Synthetases Illustrate SubstrateInduced Conformational Changes and Provide Opportunities for Drug and Herbicide Design. Journal of Molecular Biology 2008, 375 (1), 217-228. 2. Adeva, M.; Souto, G.; Blanco, N.; Donapetry, C., Ammonium Metabolism In Humans. Metabolism 2012, 61 (11), 1495-1511. 3. Kinoshita, S.; Isu, S.; Kaneko, G.; Yamada, H.; Hara, T.; Itoh, Y.; Watabe, S., The Occurrence Of Eukaryotic Type III Glutamine Synthetase In The Marine Diatom Chaetoceros Compressum. Marine Genomics 2009, 2 (2), 103-111. 4. van Rooyen, J. M.; Abratt, V. R.; Belrhali, H.; Sewell, T., Crystal Structure of Type III Glutamine Synthetase: Surprising Reversal of the Inter-Ring Interface. Structure 2011, 19 (4), 471-483. 5. Masalkar, P. D.; Roberts, D. M., Glutamine Synthetase Isoforms In Nitrogen-Fixing Soybean Nodules: Distinct Oligomeric Structures And Thiol-Based Regulation. FEBS Letters 2015, 589 (2), 215-221. 6. Moreira, C.; Ramos, M.; Fernandes, P., Glutamine Synthetase Drugability beyond Its Active Site: Exploring Oligomerization Interfaces and Pockets. Molecules 2016, 21 (8), 1028. 7. Di Tommaso, L.; Franchi, G.; Park, Y. N.; Fiamengo, B.; Destro, A.; Morenghi, E.; Montorsi, M.; Torzilli, G.; Tommasini, M.; Terracciano, L.; et al., Diagnostic Value Of HSP70, Glypican 3, And Glutamine Synthetase In Hepatocellular Nodules In Cirrhosis. Hepatology 2007, 45 (3), 725-734. 8. Fleming, K. E.; Wanless, I. R., Glutamine Synthetase Expression In Activated Hepatocyte Progenitor Cells And Loss Of Hepatocellular Expression In Congestion And Cirrhosis. Liver International 2013, 33 (4), 525-534. 9. Cooper, A.; Jeitner, T., Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain. Biomolecules 2016, 6 (2), 16. 10. Robinson, S. R., Changes In The Cellular Distribution Of Glutamine Synthetase In Alzheimer's Disease. Journal of Neuroscience Research 2001, 66 (5), 972-980. 11. Olabarria, M.; Noristani, H. N.; Verkhratsky, A.; Rodríguez, J. J., Age-Dependent Decrease In Glutamine Synthetase Expression In The Hippocampal Astroglia Of The Triple Transgenic Alzheimer's Disease Mouse Model: Mechanism For Deficient Glutamatergic Transmission? Molecular Neurodegeneration 2011, 6 (1), 1-9. 12. Eid, T.; Behar, K.; Dhaher, R.; Bumanglag, A. V.; Lee, T.-S. W., Roles of Glutamine Synthetase Inhibition in Epilepsy. Neurochemical Research 2012, 37 (11), 2339-2350. 13. Hammer, J.; Alvestad, S.; Osen, K. K.; Skare, Ø.; Sonnewald, U.; Ottersen, O. P., Expression Of Glutamine Synthetase And Glutamate Dehydrogenase In The Latent Phase And Chronic Phase In The Kainate Model Of Temporal Lobe Epilepsy. Glia 2008, 56 (8), 856-868. 14. Häberle, J.; Shahbeck, N.; Ibrahim, K.; Hoffmann, G. F.; Ben-Omran, T., Natural Course Of Glutamine Synthetase Deficiency In A 3 Year Old Patient. Molecular Genetics and Metabolism 2011, 103 (1), 89-91.


20

Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15. Rosati, A.; Marconi, S.; Pollo, B.; Tomassini, A.; Lovato, L.; Maderna, E.; Maier, K.; Schwartz, A.; Rizzuto, N.; Padovani, A.; Bonetti, B., Epilepsy In Glioblastoma Multiforme: Correlation With Glutamine Synthetase Levels. Journal of Neuro-Oncology 2009, 93 (3), 319324. 16. Kim, H. S.; Kim, J.; Im, H. N.; Yoon, J. Y.; An, D. R.; Yoon, H. J.; Kim, J. Y.; Min, H. K.; Kim, S.-J.; Lee, J. Y.; et al., Structural Basis For The Inhibition Of Mycobacterium Tuberculosis L,D-Transpeptidase By Meropenem, A Drug Effective Against Extensively DrugResistant Strains. Acta Crystallographica Section D: Biological Crystallography 2013, 69 (Pt 3), 420-431. 17. Chen, C.-T.; Alyahya, K.; Gionfriddo, J. R.; Dubielzig, R. R.; Madl, J. E., Loss Of Glutamine Synthetase Immunoreactivity From The Retina In Canine Primary Glaucoma. Veterinary Ophthalmology 2008, 11 (3), 150-157. 18. Frieg, B.; Görg, B.; Homeyer, N.; Keitel, V.; Häussinger, D.; Gohlke, H., Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies. PLoS Computational Biology 2016, 12 (2), e1004693. 19. Brookes, G.; Barfoot, P., Economic Impact Of GM Crops: The Global Income And Production Effects 1996–2012. GM Crops & Food 2014, 5 (1), 65-75. 20. Evstigneeva, Z. G.; Solov'eva, N. A.; Sidel'nikova, L. I., Methionine Sulfoximine and Phosphinothrycin: A Review of Their Herbicidal Activity and Effects on Glutamine Synthetase. Applied Biochemistry and Microbiology 2003, 39 (6), 539-543. 21. Mowbray, S.; Kathiravan, M.; Pandey, A.; Odell, L., Inhibition of Glutamine Synthetase: A Potential Drug Target in Mycobacterium tuberculosis. Molecules 2014, 19 (9), 13161. 22. Couturier, C.; Silve, S.; Morales, R.; Pessegue, B.; Llopart, S.; Nair, A.; Bauer, A.; Scheiper, B.; Poverlein, C.; Ganzhorn, A.; et al., Nanomolar Inhibitors Of Mycobacterium Tuberculosis Glutamine Synthetase 1: Synthesis, Biological Evaluation And X-Ray Crystallographic Studies. Bioorg Med Chem Lett 2015, 25(7):1455-1459. 23. Gising, J.; Nilsson, M. T.; Odell, L. R.; Yahiaoui, S.; Lindh, M.; Iyer, H.; Sinha, A. M.; Srinivasa, B. R.; Larhed, M.; Mowbray, S. L.; Karlén, A., Trisubstituted Imidazoles as Mycobacterium tuberculosis Glutamine Synthetase Inhibitors. Journal of Medicinal Chemistry 2012, 55 (6), 2894-2898. 24. Gxoyiya, B. S. B.; Kaye, P. T.; Kenyon, C., Benzimidazole-Derived ATP Analogues as Potential Glutamine Synthetase Inhibitors. Synthetic Communications 2010, 40 (17), 2578-2587. 25. Nilsson, M. T.; Krajewski, W. W.; Yellagunda, S.; Prabhumurthy, S.; Chamarahally, G. N.; Siddamadappa, C.; Srinivasa, B. R.; Yahiaoui, S.; Larhed, M.; Karlén, A.; et al., Structural Basis for the Inhibition of Mycobacterium tuberculosis Glutamine Synthetase by Novel ATPCompetitive Inhibitors. Journal of Molecular Biology 2009, 393 (2), 504-513. 26. Odell, L. R.; Nilsson, M. T.; Gising, J.; Lagerlund, O.; Muthas, D.; Nordqvist, A.; Karlén, A.; Larhed, M., Functionalized 3-amino-imidazo[1,2-a]pyridines: A Novel Class Of Drug-Like Mycobacterium Tuberculosis Glutamine Synthetase Inhibitors. Bioorganic & Medicinal Chemistry Letters 2009, 19 (16), 4790-4793. 27. Salisu, S.; Kenyon, C.; Kaye, P. T., Studies Towards the Synthesis of ATP Analogs as Potential Glutamine Synthetase Inhibitors. Synthetic Communications 2011, 41 (15), 2216-2225. 28. Singh, U.; Panchanadikar, V.; Sarkar, D., Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors. Journal of Biomolecular Screening 2005, 10 (7), 725-729.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 30

29. Gill, H. S.; Eisenberg, D., The Crystal Structure of Phosphinothricin in the Active Site of Glutamine Synthetase Illuminates the Mechanism of Enzymatic Inhibition. Biochemistry 2001, 40 (7), 1903-1912. 30. Nordqvist, A.; Nilsson, M. T.; Lagerlund, O.; Muthas, D.; Gising, J.; Yahiaoui, S.; Odell, L. R.; Srinivasa, B. R.; Larhed, M.; Mowbray, S. L.; Karlen, A., Synthesis, biological evaluation and X-ray crystallographic studies of imidazo[1,2-a]pyridine-based Mycobacterium tuberculosis glutamine synthetase inhibitors. MedChemComm 2012, 3 (5), 620-626. 31. Berlicki, Ł.; Kafarski, P., Computer-Aided Analysis Of The Interactions Of Glutamine Synthetase With Its Inhibitors. Bioorganic & Medicinal Chemistry 2006, 14 (13), 4578-4585. 32. Berlicki, L., Inhibitors of Glutamine Synthetase and their Potential Application in Medicine. Mini-Reviews in Medicinal Chemistry 2008, 8 (9), 869-878. 33. Wedler, F. C.; Horn, B. R., Catalytic Mechanisms Of Glutamine Synthetase Enzymes. Studies With Analogs Of Possible Intermediates And Transition States. Journal of Biological Chemistry 1976, 251 (23), 7530-7538. 34. Gill, H. S.; Pfluegl, G. M.; Eisenberg, D., Multicopy Crystallographic Refinement Of A Relaxed Glutamine Synthetase From Mycobacterium Tuberculosis Highlights Flexible Loops In The Enzymatic Mechanism And Its Regulation. Biochemistry 2002, 41 (31), 9863-72. 35. Krajewski, W.; Jones, T. A.; Mowbray, S. L., Structure Of Mycobacterium Tuberculosis Glutamine Synthetase In Complex With A Transition-State Mimic Provides Functional Insights. Proceedings of the National Academy of Sciences of the United States of America 2005, 102 (30), 10499-10504. 36. Rhee, S. G.; Chock, P. B., Mechanistic Studies Of Glutamine Synthetase From Escherichia Coli: Kinetic Evidence For Two Reaction Intermediates In Biosynthetic Reaction. Proceedings of the National Academy of Sciences of the United States of America 1976, 73 (2), 476-480. 37. Harth, G.; Clemens, D. L.; Horwitz, M. A., Glutamine Synthetase Of Mycobacterium Tuberculosis: Extracellular Release And Characterization Of Its Enzymatic Activity. Proceedings of the National Academy of Sciences of the United States of America 1994, 91 (20), 9342-9346. 38. Dhalla, A. M.; Li, B.; Alibhai, M. F.; Yost, K. J.; Hemmingsen, J. M.; Atkins, W. M.; Schineller, J.; Villafranca, J. J., Regeneration Of Catalytic Activity Of Glutamine Synthetase Mutants By Chemical Activation: Exploration Of The Role Of Arginines 339 And 359 In Activity. Protein Science : A Publication of the Protein Society 1994, 3 (3), 476-481. 39. Yadav, S. K., Computational Structural Analysis and Kinetic Studies of a Cytosolic Glutamine Synthetase from Camellia sinensis (L.) O. Kuntze. The Protein Journal 2009, 28 (9), 428. 40. Moreira, C.; Ramos, M. J.; Fernandes, P. A., Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations. Chemistry – A European Journal 2016, 22 (27), 9218-9225. 41. Issoglio, F. M.; Campolo, N.; Zeida, A.; Grune, T.; Radi, R.; Estrin, D. A.; Bartesaghi, S., Exploring the Catalytic Mechanism of Human Glutamine Synthetase by Computer Simulations. Biochemistry 2016, 55 (42), 5907–5916. 42. Maurizi, M. R.; Pinkofsky, H. B.; McFarland, P. J.; Ginsburg, A., Mg2+ Is Bound To Glutamine Synthetase Extracted From Bovine Or Ovine Brain In The Presence Of LMethionine-S-Sulfoximine Phosphate. Archives of Biochemistry and Biophysics 1986, 246 (1), 494-500.


22

Page 23 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


43. Eisenberg, D.; Gill, H. S.; Pfluegl, G. M. U.; Rotstein, S. H., Structure–Function Relationships Of Glutamine Synthetases1. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 2000, 1477 (1–2), 122-145. 44. Hanwell, M.; Curtis, D.; Lonie, D.; Vandermeersch, T.; Zurek, E.; Hutchison, G., Avogadro: An Advanced Semantic Chemical Editor, Visualization, And Analysis Platform. Journal of Cheminformatics 2012, 4 (1), 17. 45. Olsson, M. H. M.; Søndergaard, C. R.; Rostkowski, M.; Jensen, J. H., PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions. Journal of Chemical Theory and Computation 2011, 7 (2), 525-537. 46. Søndergaard, C. R.; Olsson, M. H. M.; Rostkowski, M.; Jensen, J. H., Improved Treatment of Ligands and Coupling Effects in Empirical Calculation and Rationalization of pKa Values. Journal of Chemical Theory and Computation 2011, 7 (7), 2284-2295. 47. Case, D. A.; Darden, T. A.; Cheatham, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; et al., AMBER 12. University of California, San Francisco: 2012. 48. Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C., Comparison Of Multiple Amber Force Fields And Development Of Improved Protein Backbone Parameters. Proteins: Structure, Function, and Bioinformatics 2006, 65 (3), 712-725. 49. Wickstrom, L.; Okur, A.; Simmerling, C., Evaluating the Performance of the ff99SB Force Field Based on NMR Scalar Coupling Data. Biophysical Journal 2009, 97 (3), 853-856. 50. Dennington, R.; Keith, T.; Millam, J., GaussView, Version 5. Semichem Inc., S. M., KS, Ed. 2009. 51. Svensson, M.; Humbel, S.; Froese, R. D. J.; Matsubara, T.; Sieber, S.; Morokuma, K., ONIOM: A Multilayered Integrated MO+MM Method For Geometry Optimizations And Single Point Energy Predictions. A Test For Diels-Alder Reactions And Pt(P(T-Bu)(3))(2)+H-2 Oxidative Addition. Journal of Physical Chemistry 1996, 100, 19357-19363. 52. Dapprich, S.; Komaromi, I.; Byun, K. S.; Morokuma, K.; Frisch, M. J., A New ONIOM implementation In Gaussian98. Part I. The Calculation Of Energies, Gradients, Vibrational Frequencies And Electric Field Derivatives. THEOCHEM 1999, 461, 1-21. 53. Vreven, T.; Byun, K. S.; Komaromi, I.; Dapprich, S.; Montgomery, J. A.; Morokuma, K.; Frisch, M. J., Combining Quantum Mechanics Methods With Molecular Mechanics Methods In ONIOM. J Chem Theory Comput 2006, 2 (3), 815-826. 54. Schrödinger, L., The PyMOL Molecular Graphics System, Version 1.8. The PyMOL Molecular Graphics System, Version 1.8 2015. 55. Frisch, M. J. T., G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; et al. Gaussian, Inc., Wallingford CT Gaussian 09, Revision D.01, Gaussian Inc.: 2009. 56. Lee, C.; Yang, W.; Parr, R. G., Development Of The Colle-Salvetti Correlation-Energy Formula Into A Functional Of The Electron Density. Physical Review B 1988, 37, 785-789. 57. Becke, A. D., Density-Functional Thermochemistry. III. The Role Of Exact Exchange. J Chem Phys 1993, 98, 5648. 58. Ditchfield, R.; Hehre, W. J.; Pople, J. A., Self-Consistent Molecular-Orbital Methods .9. Extended Gaussian-Type Basis For Molecular-Orbital Studies Of Organic Molecules. J. Chem. Phys. 1971, 54, 724. 59. Zhao, Y.; Truhlar, D. G., The M06 Suite Of Density Functionals For Main Group Thermochemistry, Thermochemical Kinetics, Noncovalent Interactions, Excited States, And


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 30

Transition Elements: Two New Functionals And Systematic Testing Of Four M06-Class Functionals And 12 Other Functionals. Theoretical Chemistry Accounts 2008, 120 (1-3), 215241. 60. Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H., A Consistent And Accurate Ab Initio Parametrization Of Density Functional Dispersion Correction (DFT-D) For The 94 Elements HPu. The Journal of Chemical Physics 2010, 132 (15), 154104. 61. Hirshfeld, F. L., Bonded-Atom Fragments For Describing Molecular Charge Densities. Theoretica chimica acta 1977, 44 (2), 129-138. 62. Ritchie, J. P., Electron Density Distribution Analysis For Nitromethane, Nitromethide, And Nitramide. Journal of the American Chemical Society 1985, 107 (7), 1829-1837. 63. Calixto, A. R.; Brás, N. F.; Fernandes, P. A.; Ramos, M. J., Reaction Mechanism of Human Renin Studied by Quantum Mechanics/Molecular Mechanics (QM/MM) Calculations. ACS Catalysis 2014, 4 (11), 3869-3876. 64. Unno, H.; Uchida, T.; Sugawara, H.; Kurisu, G.; Sugiyama, T.; Yamaya, T.; Sakakibara, H.; Hase, T.; Kusunoki, M., Atomic Structure of Plant Glutamine Synthetase: A Key Enzyme For Plant Productivity. Journal of Biological Chemistry 2006, 281 (39), 29287-29296.


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