Metaphosphate in the Active Site of Fructose-1,6-bisphosphatase*

Jun-Yong Choe, Cristina V. Iancu, Herbert J. Fromm, and Richard B. HonzatkoDagger

From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011

Received for publication, December 5, 2002, and in revised form, February 5, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The hydrolysis of a phosphate ester can proceed through an intermediate of metaphosphate (dissociative mechanism) or through a trigonal bipryamidal transition state (associative mechanism). Model systems in solution support the dissociative pathway, whereas most enzymologists favor an associative mechanism for enzyme-catalyzed reactions. Crystals of fructose-1,6-bisphosphatase grow from an equilibrium mixture of substrates and products at near atomic resolution (1.3 Å). At neutral pH, products of the reaction (orthophosphate and fructose 6-phosphate) bind to the active site in a manner consistent with an associative reaction pathway; however, in the presence of inhibitory concentrations of K+ (200 mM), or at pH 9.6, metaphosphate and water (or OH-) are in equilibrium with orthophosphate. Furthermore, one of the magnesium cations in the pH 9.6 complex resides in an alternative position, and suggests the possibility of metal cation migration as the 1-phosphoryl group of the substrate undergoes hydrolysis. To the best of our knowledge, the crystal structures reported here represent the first direct observation of metaphosphate in a condensed phase and may provide the structural basis for fundamental changes in the catalytic mechanism of fructose-1,6-bisphosphatase in response to pH and different metal cation activators.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphatases, mutases, kinases, and nucleases all catalyze phosphoryl transfer reactions central to biochemical processes that sustain life. The transfer of the phosphoryl group of a phosphate ester to water in most cases requires metal ions as cofactors, and proceeds either by way of a trigonal-bipyramidal transition state (associative mechanism), or through the formation of an unstable intermediate of metaphosphate (dissociative mechanism) (1-6). The metaphosphate anion (PO<UP><SUB>3</SUB><SUP>−</SUP></UP>) was first proposed as an intermediate in the hydrolysis of phosphate esters nearly 50 years ago (7, 8). Although it exists as a stable entity in the gas phase, where it is relatively non-reactive (9), metaphosphate is unstable in aqueous solutions, and its existence is inferred only by indirect evidence (9-15). The likelihood of trapping metaphosphate in the active site of an enzyme is remote, because of the proximity of acceptor molecules in the active site. Yet an enzyme offers an advantage in that it reduces the free energy of the transition state. Hence, the active site itself could serve as a thermodynamic trap if metaphosphate, once generated, is denied access to an acceptor.

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11, hereafter FBPase)1 is a key regulatory enzyme in gluconeogenesis. FBPase catalyzes the hydrolysis of fructose 1,6-bisphosphate (F16P2) to fructose 6-phosphate (F6P) and orthophosphate (Pi). The inhibition of FBPase in mammals results in reduced levels of serum glucose in the fasting state. Hence, FBPase is a target for the development of drugs in the treatment of non-insulin dependent diabetes, which afflicts over 15 million people in the United States (16, 17).

FBPase can be in either of two quaternary conformations, the R-state (catalytically active) or the T-state (inactive) (18). AMP and fructose 2,6-bisphosphate both inhibit catalysis by FBPase, the former through an allosteric mechanism (19, 20) and the latter by direct ligation of the active site (21, 22). Divalent metals (Mg2+, Mn2+, or Zn2+) are essential for FBPase activity. Monovalent metals (K+, Rb+, Tl+, or NH3+) further enhance reaction rates at relatively low concentrations, but can be inhibitory at high concentrations (23-25). The enzyme-mediated reaction is pH-dependent; plots of initial velocity versus Mg2+ are sigmoidal (Hill coefficient of 2) at neutral pH, but hyperbolic at pH 9.6 (26). The kinetic mechanism at pH 7 with Mg2+ as the cation activator is steady-state random (25), whereas at pH 9.6 the kinetic mechanism is rapid-equilibrium random (26). The catalytic mechanism is also sensitive to the type of cation activator: The Mn2+-activated enzyme uses exclusively the alpha -anomer of F16P2 (27), but Mg2+-activated FBPase uses both alpha - and beta -anomers of F16P2 (23). Mn2+-activated FBPase, but not the Mg2+-activated enzyme, hydrolyzes the substrate analogue (Sp)-[1-18O]fructose 1-phosphothioate 6-phosphate (28). The zinc cation is an activator of FBPase, and yet traces of Zn2+ reduce catalytic rates of the Mg2+-activated enzyme (24). The above suggests alternative catalytic pathways, the dominant mechanism being determined by pH, the kind of cation-activator, and the conformation of the substrate.

FBPase crystallizes readily from an equilibrium mixture of products and substrates, and in fact the enzyme itself is active under conditions of crystallization. In past crystal structures of FBPase, only orthophosphate and F6P have appeared in the active site (29, 30). Presumably, the observed complexes represent a minimum free energy under the conditions of the crystallization experiment. Reported here are crystal structures of FBPase at near atomic resolution, in which a partial reaction (essentially the second step of a dissociative pathway) has lead to the formation of metaphosphate in the active site. The crystallographic structures do not constitute irrefutable evidence of a dissociative reaction pathway, but they do demonstrate that FBPase can generate and stabilize metaphosphate at its active site. Hence, variations observed in the mechanism of catalysis by FBPase may arise from changes in the rate-limiting step of a dissociative pathway or even a change in pathway, for instance, from associative to dissociative.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Protein Isolation and Crystallization-- FBPase was isolated as described previously (29-31). Crystals in 10 or 200 mM K+ grew (by the method of hanging drops) from equal parts of a protein solution (10 mg/ml FBPase, 50 mM Hepes, 5 mM MgCl2, 5 mM F16P2, 10 mM or 200 mM KCl) and a precipitant solution (100 mM Hepes pH 7, 5% t-butyl alcohol, 27 or 21% (v/v) of glycerol, and 8% (for 10 mM KCl) or 14% (for 200 mM KCl) (w/v) polyethylene glycol 3350). Crystals at pH 9.6 grew from equal parts of a protein solution (10 mg/ml FBPase, 10 mM KPi, 5 mM MgCl2, 5 mM F16P2), and a precipitant solution (100 mM glycine, pH 9.6, 5% t-butyl alcohol, 25% (v/v) glycerol, and 10% (w/v) polyethylene glycol 3350). The droplet volume was 4 µl. Wells contained 500 µl of the precipitant solution. Crystals of uniform dimension (0.2 mm) grew in three to 5 days at room temperature. All crystals belong to space group I222, with one subunit of FBPase in the asymmetric unit.

Data Collection-- Data from the low K+ complex (control complex) were collected at APS-Structural Biology Center (beam line 19BM), Argonne National Laboratory, on an SBC-CCD at 100 K, using a wavelength of 1.0 Å. Data from the high K+ complex were collected at the synchrotron beam line X4A, Brookhaven National Laboratory, on an ADSC Quantum4 CCD at 100 K, using a wavelength of 0.9795 Å. Data from the high pH complex were collected at APS-BioCars (beam line 14BM), Argonne National Laboratory on an ADSC Quantum4 CCD at 100 K, using a wavelength of 1.0 Å. Data from synchrotron sources were reduced and scaled by Denzo/Scalepack (32).

Structure Determination and Refinement-- Crystals grown for this study are isomorphous to PDB code 1EYJ. Structure determinations were initiated by molecular replacement using calculated phases of 1EYJ, and then refined by SHELX (33). Model building and modifications employed XTALVIEW (34). Only distance restraints between covalently link atoms were applied to orthophosphate and metaphosphate, allowing structure factors to be the principal determinant of the geometry of the phosphoryl groups in each of the complexes. Fractional occupancy factors for a given atom or group of atoms were determined by refinement of thermal parameters for a series of fixed occupancy factors. Reported here are occupancy factors that resulted in refined thermal parameters comparable to those of neighboring atoms in full occupancy.

    RESULTS
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Three complexes (Table I) are presented here: (i) orthophosphate, F6P, Mg2+, and K+ (10 mM) at pH 7 (hereafter the control complex), (ii) metaphosphate, F6P, Mg2+, and K+ (200 mM) at pH 7 (hereafter the high K+ complex), and (iii) metaphosphate, F6P, and Mg2+ at pH 9.6 (hereafter the high pH complex). All complexes have the dynamic loop (residues 52-72) in its engaged conformation, as determined by the location of Tyr57 (29, 30). Conditions that result in the formation of metaphosphate differ only in pH (7 versus 9.6) or the concentration of K+ (10 versus 200 mM). The kinetic mechanism of FBPase changes from steady-state random at pH 7 to rapid-equilibrium random at pH 9.6 (26), and K+ is inhibitory at concentrations of 200 mM (Ki of 68 mM), but activating at concentrations of 10 mM (Ka of 17 mM) (25).


                              
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Table I
Statistics of data collection and refinement

Control Complex (PDB: 1NUY)-- Aside from its substantially higher resolution (Table I), the control complex is essentially identical to the Mg2+ product complex of Choe et al. (29, 30, 40). Orthophosphate is clearly at the active site, and Mg2+ occupies sites 1, 2, and 3 (Fig. 1A). The 1-OH group of F6P is in two positions, related by a rotation of approximately of 120° about the C1-C2 bond axis. In one of its positions (occupancy factor of 0.7), the 1-OH group coordinates the Mg2+ at site 1, optimally positioned for an associative reaction (Fig. 2A). Three of four oxygen atoms from Pi are approximately equidistant from the 1-OH group of F6P and the distance between the phosphorus atom and the oxygen atom of the 1-OH group is 2.72 Å. In its second position (displaced conformation; occupancy factor of 0.3), the 1-OH group is turned away from in-line geometry, hydrogen bonding with only one of the oxygen atoms of Pi (Fig. 2A). Mg2+ at site 1 is 4-5 coordinated (the 1-OH group of F6P being responsible for the variation), whereas magnesium cations at sites 2 and 3 are both six-coordinated (Fig. 2A).


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Fig. 1.   Stereoviews of FBPase active sites. A, complex of Mg2+, F6P, and orthophosphate at pH 7 (control complex). B, complex of Mg2+, K+/Zn2+, F6P, and metaphosphate at pH 7 (high K+ complex). C, complex of Mg2+, F6P, and metaphosphate at pH 9.6 (high pH complex). Electron density (blue) covers metaphosphate/hydroxide anions or orthophosphate at a contour level of 3 sigma , using a cutoff radius of 1 Å. Anomalous difference density (red) covers site M1 at a contour level of 3 sigma , using a cutoff radius of 1 Å. MOLSCRIPT (39) and RASTER3D (35) were used for the illustration.


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Fig. 2.   Distance relations between selected atoms in the active site. A, control complex. B, high K+ complex. C, high pH complex. The dotted outline in panel C represents a channel of electron density that extends between metal sites 3 and 4, which in the refined model is represented by a discrete set of water molecules and magnesium cations.

High K+ Complex (PDB: 1NUX)-- An increase in the concentration of K+ from 10 to 200 mM results in an anomalous signal at metal site 1 (Fig. 1B), indicating the displacement of the Mg2+ from that site by a different atom type. The metal at site 1 has four inner-sphere ligands: One oxygen atom each from Asp118, Aspl21, Glu280, and the phosphoryl species is ~2-2.5 Å from metal site 1 (Fig. 2B). The 1-OH group of F6P no longer coordinates the site-1 metal, being entirely in its displaced conformation. The anomalous signal from metal site 1 may be due to K+ or a trace contamination of a transition series cation (Zn2+, for instance) in the KCl. The latter is the most likely cause. The inclusion of 0.2 mM EDTA in the crystallization solution eliminates the anomalous signal at metal site 1. Furthermore, the tetrahedral coordination of the site-1 cation and the displaced conformation of the 1-OH group of F6P are characteristics of previously determined Zn2+-product complexes of FBPase (29, 30). A mixture of Zn2+ and Mg2+ at occupancies of 0.25 and 0.75, respectively, account for the magnitude of the anomalous signal, and provide thermal parameters of ~24 Å2, equivalent to that of the site-1 Mg2+ in the control structure

The thermal parameter associated with the Mg2+ at site 3 is higher than that of its counterpart in the control complex (30 versus 26 Å2). The Mg2+ cation may not fully occupy site 3. (As defined more clearly in the high pH complex below, Mg2+ could occupy a site near Glu98 at low occupancy, and not be resolved from the electron density associated with the water molecule that hydrogen bonds with Glu98 and coordinates the Mg2+ at site 3. See Fig. 2, B and C.) Further indications of weakened interactions involving Mg2+ at site 3 are an increase in the coordination distance to the oxygen atom of the phosphoryl species and the concomitant decrease in the donor-acceptor distance to Arg276 of that same oxygen atom (Fig. 2B).

The electron density associated with the ligand at the 1-phosphoryl pocket is a distorted tetrahedron. An elongated teardrop of electron density extends from a plane of electron density. Metaphosphate fits the planar density well, and a water molecule (perhaps representing a molecule of hydroxide) fits equally well to the teardrop of electron density. Thermal parameters of the metaphosphate molecule and its associated water/hydroxide molecule, both refined at full occupancy, are comparable to those of the ligating Mg2+ and nearby side chains. The water molecule is 2.35 Å away from the phosphorus atom of metaphosphate, being coordinated to the magnesium cations at sites 2 and 3, and is on the verge of hydrogen bonding to the side chain of Asp74 (Fig. 2B). The electron density probably represents an equilibrium mixture of orthophosphate and PO<UP><SUB>3</SUB><SUP>−</SUP></UP>/OH-.

High pH Complex (PDB: 1NUW)-- Glu97, which in the control and high K+ complexes coordinates magnesium cations at sites 2 and 3, now bridges the magnesium cations at sites 1 and 2. The Mg2+ at site 1 is at least 5-coordinated and the Mg2+ at site 2 remains six-coordinated (Figs. 1C and 2C). As in the high K+ complex, the 1-OH group of F6P is in its displaced orientation. The loss of Glu97 as a coordinating ligand to Mg2+ at site 3 is linked perhaps to the binding of magnesium cations at sites 3 and 4. A similar conformational change in Glu97 occurs in Mg2+/Tl complexes, in which Tl+ at site 4 displaces Mg2+ at site 3 (40). Glu98 coordinates to Mg2+ at site 4, whereas Asp68 coordinates to Mg2+ at site 3, but the binding of metal cations to sites 3 and 4 are probably mutually exclusive, as they are only 2.5 Å apart. Thermal parameters for Mg2+ at sites 3 and 4 are 23 and 21 Å2, respectively, with fractional occupancies of 0.6 and 0.3. The electron density at the 1-phosphoryl pocket appears as an elongated teardrop extending from a plane (Fig. 1C). A molecule of metaphosphate, distorted from planarity, and a water molecule (or hydroxide anion) provide the best fit to the electron density. The oxygen atom of the water molecule is 3.09 Å from the phosphorus atom of the metaphosphate, and is within the coordination spheres of the magnesium cations at sites 2 and 4 (or 3), and within hydrogen bonding distance of Asp74 (Fig. 2C). As in the high K+ structure, the electron density at the 1-phosphoryl pocket is consistent with an equilibrium mixture of orthophosphate and PO<UP><SUB>3</SUB><SUP>−</SUP></UP>/OH-.

    DISCUSSION
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The control complex may represent a step on the reaction pathway, but it almost certainly does not represent the central kinetic complex (the interconversion of F16P2 and F6P/Pi at the active site). Liu and Fromm (22) determined a value of 2 for the equilibrium constant of the central kinetic complex. The electron density is consistent, however, with the presence of only F6P and orthophosphate. Conditions of crystallization and packing interactions of the crystal could perturb the equilibrium constant of the central complex in favor of products, or the crystallographic complex itself could represent a dead-end complex not on the reaction pathway. Nonetheless, the geometric relationship between Pi and F6P (Fig. 2A) is consistent with an associative reaction mechanism, the details of which have been presented elsewhere (40); Asp74 (the pKa of which is raised by the proximity of Glu98) abstracts a proton from a water molecule coordinated to the Mg2+ at site 2 or site 3. The resulting Mg2+-coordinated hydroxide anion in turn abstracts the proton from a second water molecule (the attacking nucleophile) that bridges the magnesium cations at sites 2 and 3. The attacking hydroxide anion has in-line geometry with respect to the 1-phosphoryl group, and would be less than 3 Å from the P-1 atom. Data in support of catalytic roles for Asp74 and Glu98 come from directed mutations, which cause more than a 10,000-fold reduction in catalytic rates (31, 36). The associative pathway must have a double proton transfer, because the catalytic base (Asp74/Glu98 subassembly) is too far from the water molecule bridging the magnesium cations at sites 2 and 3 for a direct hydrogen bond.

The arrangement of catalytic side chains and ligands in the active site of FBPase, however, are also consistent with a dissociative pathway, and indeed FBPase can generate metaphosphate and the hydroxide anion from orthophosphate. To the best of our knowledge, the high pH and high K+ complexes reported here are the first direct observations of metaphosphate in a condensed phase. FBPase in its crystalline complex catalyzes the second step of a dissociative mechanism; however, the formation of F16P2 from PO<UP><SUB>3</SUB><SUP>−</SUP></UP> and F6P (the first step of a dissociative mechanism) may be inaccessible because of the mutual rotation of the plane of metaphosphate and the 1-OH group of F6P away from in-line geometry (Fig. 1, B and C). Evidently, the crystalline complex is dead-end with respect to the overall reaction, but clearly the active site of FBPase can at least equalize the free energies of bound meta- and orthophosphate. Variations in the kinetic and/or catalytic mechanisms due to changes in pH, chemical composition and/or conformation of the substrate, and to the type of metal activator (23-28), may stem from differences in the rate-limiting step of a dissociative pathway and/or a change in the type of pathway (dissociative versus associative).

The presence of Mg2+ cations at mutually exclusive loci (sites 3 and 4) and the alternative ligation of cation sites by Glu97 suggest the possibility of significant change in the active site during the course of the reaction. The Mg2+ at site 4, a cation-binding site identified in Mg2+/Tl+ complexes of FBPase (40), may in fact play a direct role in catalysis (Fig. 3). In F16P2 complexes of FBPase, Mg2+ may appear initially at sites 1, 2, and 4. Stereoinversion of the 1-phosphoryl group may favor the migration of Mg2+ from site 4 to 3. Hence, in all crystalline complexes of Pi and F6P, Mg2+ (or Zn2+) is at site 3, whereas in F16P2 complexes the preferred binding site for Mg2+ may be site 4. Asp74 then could activate a water molecule coordinated to magnesium cations at sites 2 and 4 for a nucleophilic attack on the metaphosphate anion (Fig. 3).


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Fig. 3.   Possible dissociative reaction pathway for FBPase. The coordination spheres of metal cations (M1 through M4) are not shown in full. The Mg2+ at site 2 is six-coordinated, and the coordinating ligands do not change over the proposed reaction pathway. A, initial substrate complex. Mg2+ at site M1 is six-coordinated. As PO<UP><SUB>3</SUB><SUP>−</SUP></UP> forms, negative charge builds on the O-1 atom of F16P2, and Glu97 leaves the coordination sphere of M1. B, PO<UP><SUB>3</SUB><SUP>−</SUP></UP>/H2O complex. Metaphosphate is present, and Asp74 abstracts a proton from the water molecule bound to the Mg2+ at site 4. The events of panel B may precede those of panel A, or occur in synchrony with those of panel A. C, PO<UP><SUB>3</SUB><SUP>−</SUP></UP>/OH- complex. Mg2+ migrates from site 4 to site 3. D, product complex. The crystal structure of the control complex represents the end-state of the reaction.

A change in metal cation coordination by Glu97 could facilitate the putative migration of Mg2+ from site 4 to 3 during catalysis. In a dissociative pathway, charge builds on the O-1 atom of F16P2, and as a consequence the Mg2+ at site 1 may release Glu97 from its crowded coordination sphere. A modest conformational change puts Glu97 into the nascent coordination sphere of metal site 3, which in turn promotes the migration of the cation (with its attached hydroxide anion) from site 4 to 3 (Fig. 3). The migration of Mg2+ between sites 3 and 4 would account for the significance of both Asp68 (which coordinates Mg2+ at site 3) and Glu98 (which coordinates Mg2+ at site 4) in catalysis. Mutations2 of Glu98 (36) and Asp68 each reduce catalytic rates of FBPase by orders of magnitude under comparable conditions of assay. The dissociative pathway, as in Fig. 3, replaces the double proton transfer of the associative pathway, with the migration of a metal-bound hydroxide anion.

The loss of Mg2+ cooperativity at pH 9.6 in FBPase kinetics may simply reflect a change in the rate-limiting step of a dissociative pathway. Mutations of Asp68 eliminate Mg2+ cooperativity at pH 72 and thereby implicate the cation in site 3 in cooperative phenomenon. The generation of the hydroxide anion at pH 7 may require the participation of Asp74 and metal cations at sites 2 and 4 (or 3), whereas at pH 9.6 the generation of the hydroxide anion may occur with less involvement from the active site. Hence, the formation of metaphosphate may be limiting at pH 9.6, whereas the formation of hydroxide anion may be limiting at pH 7.

The hydrolysis of most phosphate esters in organic model systems occurs by a dissociative mechanism (3-6), and the same chemistry may occur in the active site of FBPase. In fact, Herschlag and Jencks (37) present a scheme (Chart II or III) that is strikingly similar to the complexes reported here in the relative placement of Mg2+, a metal-coordinated hydroxide ion and a planar intermediate. Furthermore, there are striking parallels between the active sites of FBPase and alkaline phosphatase (38), suggesting that the chemistry of model systems is broadly applicable to phosphatases. Ancestral phosphatases long ago may have commandeered the dominant reaction pathway in solution, but through evolution the free-energy landscape of the reaction coordinate may now place the associative and dissociative pathways on a near equal footing. Hence, small perturbations in the relative free energies of transition states, due to changes in metal cofactors, pH, chemical composition of the substrate, and/or conditions of crystallization may leverage profound effects on the catalytic mechanism.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Research Grant NS 10546 and National Science Foundation Grant MCB-9985565.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1NUX, 1NUY, and 1NUW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Dagger To whom correspondence should be addressed. Tel.: 515-294-7103; Fax: 515-294-0453; E-mail: honzatko@iastate.edu.

Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M212395200

2 C. V. Inacu and R. B. Honzatko, unpublished data.

    ABBREVIATIONS

The abbreviations used are: FBPase, fructose-1,6-bisphosphatase; F16P2, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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