Allosteric Communication of Tryptophan Synthase

FUNCTIONAL AND REGULATORY PROPERTIES OF THE beta S178P MUTANT*

Anna MarabottiDagger , Daniela De Biase§, Angela Tramonti§, Stefano BettatiDagger , and Andrea MozzarelliDagger ||

From the Dagger  Institute of Biochemical Sciences and  National Institute for the Physics of Matter, University of Parma, 43100 Parma, Italy and the § Department of Biochemical Sciences "A. Rossi Fanelli," University of Rome "La Sapienza," 00185 Rome, Italy

Received for publication, December 29, 2000, and in revised form, February 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The alpha 2beta 2 tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the beta S178P mutant. Ser-178 is located at the end of helix 6 of the beta  subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of beta Ser-178 is hydrogen bonded to Gly-181 of loop 6 of the alpha  subunit only when alpha  subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the beta S178P mutation suggests that the hydrogen bond involving alpha Gly-181 is disrupted as a result of localized structural perturbations. The ratio of alpha  to beta  subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight alpha -beta complex. Both the activity of the alpha  subunit and the inhibitory effect of the alpha  subunit ligands indole-3-acetylglycine and D,L-alpha -glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the beta  subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and D,L-alpha -glycerol-3-phosphate do not affect the beta  activity. Accordingly, the distribution of L-serine intermediates at the beta -site, dominated by the alpha -aminoacrylate, is only slightly influenced by alpha  subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the alpha -aminoacrylate tautomer absorbing at 450 nm. Crystals of the beta S178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the alpha  and beta  activity in the alpha 2beta 2 complex with respect to the isolated subunits results from interactions that involve residues different from beta Ser-178 and (ii) beta Ser-178 is a critical residue in ligand-triggered signals between alpha  and beta  active sites.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Allosteric proteins are key elements of cellular regulation, because they allow for a fine adaptation of the living organisms to changes in metabolite concentration and physico-chemical conditions. Tryptophan synthase is an allosteric enzyme, composed of two alpha  and two beta  subunits, catalyzing the last two steps in the biosynthesis of L-tryptophan (1-3) (Fig. 1). The alpha  active site cleaves indole-3-glycerol phosphate into indole and D-glyceraldehyde-3-phosphate. The beta  active site contains a pyridoxal 5'-phosphate molecule bound to Lys-87 via a Schiff base and catalyzes the replacement of the beta -hydroxyl of L-serine with indole. The latter compound is intramolecularly channeled from the alpha  site via a hydrophobic tunnel connecting the alpha  and beta  active sites, as proposed on the basis of the first crystal structure of the enzyme (4). The alpha  and beta  activities are reciprocally modulated. Extensive functional and structural studies of the wild type enzyme and several mutants have unveiled some of the mechanisms underlying catalysis and allosteric regulation (3). In particular, the beta  subunit exists either in a closed, catalytically active state, when the alpha -aminoacrylate is the most populated catalytic intermediate, or in an open, catalytically less active state, when the external aldimine is the predominant species (5-8). When the former intermediate is formed at the beta  active site, signals are generated that trigger the activation of the alpha  active site, keeping in phase the action of the two sites. Intersubunit communications are mediated by a movable domain of the beta  subunit, called the COMM1 (communication) domain, interacting with the mobile loops 2 and 6 of the alpha  subunit (9, 10). By comparing the three-dimensional structure of the enzyme in the presence and absence of the alpha  subunit ligand 5-fluoroindole-3-propanol phosphate and the alpha -aminoacrylate species (10), it was noted that six new polar interactions are formed, five of which are between residues of alpha  loop 2 and beta  helix 6, and only one is between alpha Gly-181 of loop 6 and beta Ser-178 of helix 6. Whether there is a unique pathway or multiple specialized pathways of intersubunit communication is not yet known. In fact, the subunit interface mediates i) the 10-100-fold activation of the alpha  and beta  activity in the alpha 2beta 2 complex with respect to the isolated subunits, ii) the reciprocal action of alpha  and beta  subunit ligands on kinetic, thermodynamic, and dynamic properties of the opposite active sites, and iii) pH and monovalent cation modulation of beta  subunit activity.


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Fig. 1.   Top panel, three-dimensional structure of the alpha /beta dimer of the tryptophan synthase from the file 1a50.pdb (10). The alpha  subunit is on the left (light gray), and the beta  subunit is on the right (dark gray). The coenzyme pyridoxal 5'-phosphate and the alpha  subunit inhibitor 5-fluoroindole-3-propanol phosphate are shown in ball-and-stick mode. The figure was prepared using the program WebLab Viewer Pro 3.20 (Molecular Simulations Inc.). Bottom panel, scheme of the alpha  and beta  reactions of the tryptophan synthase.

To gain insight into the role of beta Ser-178-alpha Gly-181 interaction in regulating the allosteric communication, beta Ser-178 was mutated to a proline residue. The rationale for the choice of proline was that the interaction between beta Ser-178 and alpha Gly-181 is mediated by a hydrogen bond between the carbonyl oxygen of beta Ser-178 and the amidic nitrogen of alpha Gly-181. beta Ser-178 is not a conserved residue, as demonstrated by a multiple alignment of the amino acid sequence of beta  subunits from different sources.2 Therefore, proline mutation was chosen for its unique feature of preventing the formation of the hydrogen bond with alpha Gly-181.3 Furthermore, to obtain structure and function relationships, crystals of the mutant were grown, and the catalytic competence of the mutant was investigated by polarized absorption microspectrophotometry.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- All chemicals, obtained from Sigma, except cesium chloride (Roche Molecular Biochemicals), were of the best available quality and were used without further purification. Glyceraldehyde-3-phosphate was obtained from the monobarium salt of the diethyl acetal form, and its concentration was determined using an assay based on the activity of glyceraldehyde-3-phosphate dehydrogenase. The stock solution was kept at pH <2 and stored at -20 °C. Oligonucleotides were from Eurogentec. Restriction enzymes and the agarose gel extraction kit were from Roche Molecular Biochemicals. Ligation was performed by using a DNA ligation system from Amersham Pharmacia Biotech.

alpha and beta  Activity Assays-- The beta  activity assay was carried out as previously described (11). The buffer for the assay was stored at -20 °C, kept in the dark, and used within 15 days from preparation. The assay for the alpha  subunit reverse activity was carried out in 50 mM bis-Tris propane, 0.2 mM indole, 0.04 mM pyridoxal 5'-phosphate, and 0.5 mM glyceraldehyde-3-phosphate, pH 7.8, at 20 °C. The assay solution was prepared immediately before use and kept at 4 °C. Measurements were terminated within 1 h from solution preparation. Activity assays were carried out using 1-mm path length quartz cuvettes.

Spectrophotometric and Spectrofluorimetric Measurements-- Measurements were carried out in a solution containing 25 mM bis-Tris propane in the absence and presence of 50 mM L-serine at 20 °C. The final pH was adjusted with concentrated HCl. pH measurements were performed with a Radiometer pHM83 pH meter, equipped with an Ingold-Mettler Lot406-M3 microelectrode. The effect of pH on the equilibrium distribution of catalytic intermediates was measured in a solution containing 50 mM MOPS, 50 mM Bicine, 50 mM proline, and 1 mM EDTA (MBP buffer). The pH was raised to 11.5 with concentrated sodium hydroxide and then back-titrated with HCl to the desired pH to keep constant the ionic strength of the buffer in the pH range 6-11. Spectrophotometric measurements were carried out using a Varian Cary400. Spectrofluorimetric measurements were carried out using a PerkinElmer LS50B. Data were analyzed with the software SigmaPlot 6.0 (SPSS Science).

Computational Methods-- Molecular simulations of the site-directed mutagenesis were carried out using SYBYL version 6.5 (Tripos Associates). The Tripos force field was used throughout the calculations. The Ser to Pro mutation was carried out using the Biopolymer option on the tryptophan synthase structure with 5-fluoroindole-3-propanol phosphate bound to the alpha -subunit, Protein Data Bank file 1a50.pdb (10). Charges were assigned according to Gasteiger-Huckel, and water molecules present in the Protein Data Bank file were included in the calculations. A first minimization was performed on the mutated amino acid to reduce negative contacts with neighboring residues, and then a second minimization was performed on the alpha /beta interface of the enzyme. The radius of the latter minimized region was 10 Å, centered at the mutation site when all interactions were considered, and 12 Å when only steric interactions were considered. The remaining portion of the protein was kept rigid. The minimizations were performed according to the Powell method. The resulting structure was superimposed on the wild type for evaluating structural changes upon mutation.

Preparation of the beta S178P Mutant-- The bacterial strain Escherichia coli CB149 carrying the plasmid pEBA10 for the expression of tryptophan synthase complex from Salmonella typhimurium was kindly provided by Dr. E. W. Miles, National Institutes of Health. Site-directed mutagenesis was performed by overlap extension PCR (12). External primers annealing over the 5'- and 3'-terminal sequences of the trpB gene in the pEBA10 plasmid were PE1 and PE2, respectively, as described by Yang et al. (13). Mutagenic primers for beta S178P were 5'-CGTAACTACCGGGCCAGTCGC-3' and its complementary sequence (S178Prev and S178Pfrw, respectively). PCR was performed at 95 °C for 50 s, 62 °C for 50 s, and 74 °C for 60 s, with 2.5 units of Vent polymerase, using plasmid pEBA10 as template. The 570- and 810-base pair products from the first PCR reaction were used as templates in a second PCR with the external primers PE1 and PE2 to generate the complete coding sequence of trpB. The 1100-base pair BglII/XbaI fragment generated from the second PCR was subcloned into pEBA1 and digested with the same restriction enzymes. The newly generated plasmid was used to transform E. coli JM109 and then E. coli CB149. The trpB gene carrying the mutation S178P was fully sequenced. Sequence analysis showed the presence of the correct S178P mutation, as well as an Arg to Ser mutation at position 34 of the amino acid sequence. This mutation was already present in the original plasmid.4

Purification of the beta S178P Mutant-- The growth of the bacterial strain E. coli CB149 containing the plasmid pEBA10, with the mutation S178P in the trpb gene, of the S. typhimurium tryptophan synthase was carried out according to Yang et al. (13). Purification of the mutant was carried out as described for the wild type (13). Enzyme purity was checked by SDS-polyacrylamide gel electrophoresis, and the relative amount of alpha  and beta  subunits was estimated with the program Quantity One 4.0.3 (Bio-Rad). The yield of bacteria growth for the mutant was 1.5 g per 100 ml of culture, about 25% less than the yield for the wild type under our experimental conditions. The amount of purified mutant was 5 mg per g of bacteria, about 10-fold less than for the wild type. The purified enzyme was dialyzed against 50 mM Bicine, pH 7.8, and stored at -80 °C. An aliquot of enzyme solution was extensively dialyzed against 25 mM bis-Tris propane, 10 µM pyridoxal 5'-phosphate, pH 7.8, to remove monovalent cations and stored at -80 °C. Protein concentration was estimated on the basis of the absorption intensity at 280 nm (14).

Crystallization of beta S178P Tryptophan Synthase-- Crystals of the mutant were grown according to Schneider et al. (10). The mutant was crystallized using the hanging drop method by mixing 5 µl of a solution containing 10 mg/ml enzyme, 50 mM Bicine, pH 7.8, with an equal quantity of reservoir solution containing 50 mM Bicine, 1 mM EDTA, 12% (w/v) polyethylene glycol 8000, and 1.4 mM spermine, pH 7.8, at 21 °C. Monoclinic crystals grew within a few days and were stored in a solution containing 50 mM Bicine, 1 mM EDTA, and 20% polyethylene glycol 8000, at 4 °C.

Microspectrophotometric Measurements-- Single crystals of beta S178P tryptophan synthase were suspended in a solution containing 20% polyethylene glycol 8000, 50 mM bis-Tris propane, pH 7.8, and mounted in a quartz flow cell. Replacement of the suspending medium was carried out by passing solutions through the cell. The cell was placed on the stage of a Zeiss MPM03 microspectrophotometer, equipped with a 10 × Zeiss UV-visible ultrafluar objective (15, 16). Polarized absorption spectra were collected between 315 and 550 nm with the electric vector of the linearly polarized light parallel to the extinction directions on the (210) flat face of monoclinic crystals. All experiments were carried out at 20 °C.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because the three-dimensional structure of the beta S178P mutant tryptophan synthase was not available, a molecular model was created to evaluate the effects of the mutation on the protein conformation. The model was obtained by changing beta Ser-178 into a proline in the wild type crystallographic structure and performing two energy minimizations of the resulting structure. The comparison of the wild type and mutant (Fig. 2) indicates that the mutation causes only localized structural changes, leading to the loss of the hydrogen bond between beta Ser-178 and alpha Gly-181. No other significant movements appear to take place either in the alpha  or beta  subunit. An experimental evaluation of the effect of mutation on the strength of the alpha -beta association was obtained by the quantitative densitometric analysis of the stained bands in the electrophoretic pattern of the purified mutant and wild type protein under denaturing conditions (data not shown). It was found that the ratio of alpha  and beta  subunit concentrations is 0.7, as for the wild type (17), indicating that the mutation does not appreciably affect the intersubunit affinity of the alpha 2beta 2 mutant complex.


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Fig. 2.   Superposition of wild type (light gray) and beta S178P (dark gray) tryptophan synthase structures. The mutant enzyme is a model structure obtained after Ser to Pro mutation and energy minimization. The structure of the wild type enzyme was obtained from the file 1a50.pdb (10). The dashed line indicates the hydrogen bond between the carbonyl moiety of beta Ser-178 and the amidic nitrogen of alpha Gly-181 (3.4 Å). This hydrogen bond is lost in the beta S178P mutant.

The alpha  subunit activity of the mutant was found to be very similar to that of the wild type, and the beta  subunit activity was about 2-fold lower. Furthermore, the alpha  subunit activity of the mutant was inhibited by the binding of IAG and GP (Fig. 3, a and b), as previously observed for the wild type enzyme (18). The calculated inhibition constants of IAG and GP for the mutant, assuming a single isotherm of binding, are 0.36 ± 0.04 and 12.7 ± 2.9 mM, respectively. The same value was determined for GP in the wild type, and a 3-fold higher value was found for IAG (18). These findings indicate that the mutation does not significantly affect the alpha  active site geometry and does not impair the binding of alpha  subunit ligands. It should be noted that the activity of the mutant at infinite ligand concentrations is not fully abolished, whereas it is fully abolished for the wild type. This finding might be associated with different types of inhibition, as previously observed for indole-3-propanol phosphate, competitive with respect to glyceraldehyde-3-phosphate and noncompetitive with respect to indole (19). In contrast to that observed for the wild type, no effect of IAG (18) and GP (17) on beta  subunit activity was detected (Fig. 3, c and d), indicating that this feature of the allosteric regulation between alpha  and beta  subunits is lost in the beta S178P mutant.


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Fig. 3.   Effects of alpha  ligands IAG and GP on the rate of alpha  and beta  reactions catalyzed by beta S178P and wild type tryptophan synthase. Activities in the presence of ligands are calculated as a percent of the alpha 2beta 2 complex activity in the absence of ligands for the mutant () and wild type enzyme (). Dependence of the relative specific alpha  activity on IAG (a) and GP concentrations (b) is shown. For the mutant, the dissociation constants, calculated by fitting data to a binding isotherm, are 0.36 ± 0.04 and 12.7 ± 2.9 mM for IAG and GP, respectively. For the wild type, the dissociation constants are 0.99 ± 0.28 and 11.4 ± 4.4 mM for IAG and GP, respectively. For the mutant, the percent residual activity at infinite IAG and GP concentrations is 6.2 ± 3 and 16.8 ± 5.8, respectively. For the wild type, the activity at infinite IAG and GP concentrations is fully abolished. The alpha  activity of the wild type and mutant is 6000 and 5900 units/mg, respectively. Dependence of the beta  activity on IAG (c) and GP concentrations (d) is shown. The beta  activity of the wild type and the mutant is 1168 and 504 units/mg, respectively.

A well characterized effect of the alpha -beta allosteric regulation is the change of the equilibrium distribution of catalytic intermediates at the beta  active site caused by alpha  subunit ligands (2, 5, 8, 15, 16, 20). This distribution is also affected by pH, monovalent cations, and temperature (8, 21). Specifically, alpha  subunit ligands, low pH, cesium ions, and high temperature stabilize the alpha -aminoacrylate intermediate, whereas high pH, sodium ions, and low temperature stabilize the external aldimine. Moreover, high pH and monovalent cations favor the accumulation of a quinonoid intermediate (16). To further evaluate the effect of mutation on the alpha -beta regulation, the influence of alpha  subunit ligands, monovalent cations, and pH on the equilibrium distribution of catalytic intermediates of the beta  active site was investigated. The absorption spectrum of the mutant enzyme in the absence of ligands (Fig. 4a) exhibits a typical band at 412 nm, assigned to the ketoenamine tautomer of the internal aldimine. The second absorption band at about 330 nm, assigned to the enolimine form of the internal aldimine, is more intense than in the wild type, indicating that the latter tautomer is favored in the mutant. When L-serine reacts with the mutant tryptophan synthase in the absence of monovalent cations, at room temperature, pH 7.7, the predominant species is the alpha -aminoacrylate, mainly absorbing at 350 nm (Fig. 4b), as observed for the wild type (21). When a solution of the alpha -aminoacrylate species was titrated with increasing concentrations of IAG and GP, the band at 350 nm was slightly blue-shifted, and the absorption intensity at 450 nm exhibited a small increase (Fig. 4b). This band is attributed to the ketoenamine tautomer of the alpha -aminoacrylate absorbing at 450-470 nm, as observed in O-acetylserine sulfhydrylase (22) and cystathionine beta -synthase (23, 24). This result indicates a negligible influence of IAG and GP on the equilibrium distribution of beta -intermediates. The same negligible effect was detected by monitoring the change of the fluorescence emission at 500 nm upon excitation at 420 nm (data not shown), typical of the external aldimine, the only highly fluorescent species of tryptophan synthase (25).


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Fig. 4.   a, absorption spectrum of the mutant beta S178P tryptophan synthase. The spectrum was recorded on a solution containing 0.77 mg/ml mutant enzyme, 25 mM bis-Tris propane, pH 7.8, at 20 °C. b, effect of alpha  subunit ligands on the equilibrium distribution of the catalytic intermediates formed at the beta S178P beta  active site. Absorption spectra were collected on a solution containing 0.98 mg/ml mutant enzyme, 25 mM bis-Tris propane, 50 mM L-serine, pH 7.7, at 20 °C. Spectra were recorded in the absence (---) and presence of 1.6 mM IAG (···) or 50 mM GP (--- --- ---). GP is a disodium salt.

Sodium ions strongly favor the accumulation of the external aldimine species of tryptophan synthase (21). This behavior was attributed to the stabilization of a partially open conformation of the enzyme (21, 26). Binding of sodium ions to the mutant causes a very small increase of the absorption band at 422 nm, assigned to the external aldimine (data not shown). Accordingly, the fluorescence emission at 500 nm is enhanced (Fig. 5a). The dissociation constant of sodium ions was found to be 153 mM (Fig. 5b), a value significantly higher than the dissociation constants of 1 and 19 mM previously determined for the wild type (21). IAG and GP do not significantly perturb the equilibrium distribution of intermediates also in the presence of saturating concentrations of sodium ions (data not shown).


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Fig. 5.   Effect of Na+ and Cs+ on the equilibrium distribution of the intermediates at the beta S178P beta  active site. a, the fluorescence emission spectra (lambda ex = 420 nm) were collected on a solution containing 0.1 mg/ml mutant enzyme, 25 mM bis-Tris propane, and 50 mM L-serine, pH 7.7, at 20 °C, in the absence (---) and presence of 500 mM Na+ (--- ·· ---) or 100 mM Cs+ (--- --- ---). b, fluorescence emission at 492 nm was monitored as a function of Na+ concentration. The solid curve through data points is the best fit to a binding isotherm with a dissociation constant of 153 ± 14 mM. c, fluorescence emission at 492 nm was monitored as a function of Cs+ concentration. The solid curve through data points is the best fit to a binding isotherm with a dissociation constant of 1.28 ± 0.08 mM.

Binding of cesium ions causes similar effects in the mutant and wild type enzyme. The absorption spectra recorded in the presence of cesium ions indicate a small increase of absorption at 440-460 nm (data not shown), indicating a stabilization of the ketoenamine form of the alpha -aminoacrylate, and a consistent decrease of the fluorescence emission at 500 nm (Fig. 5a). The extent of spectral changes and the calculated dissociation constant of cesium ions (Fig. 5c) are the same as for the wild type (21).

The equilibrium distribution of intermediates for the mutant enzyme in the presence of sodium ions is affected by pH (Fig. 6), as previously observed for the wild type (8). However, the pH dependence is different and is not significantly affected by GP, in contrast to that observed for the wild type. Furthermore, the amount of the external aldimine accumulated at high pH is much less than that observed in the wild type (8).


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Fig. 6.   Effect of pH on the equilibrium distribution of beta S178P beta  active site intermediates. Absorption spectra were recorded on a solution containing 0.9 mg/ml beta S178P enzyme, MBP buffer, 50 mM L-serine, 500 mM NaCl at 20 °C, at pH 6.6 (---), 8.6 (--- · ---), 9.5 (···) and 10.6 (--- --- ---). Inset, dependence on pH of the fluorescence at 500 nm (lambda ex = 420 nm) in the absence () and presence of 50 mM GP ().

In the wild type enzyme the reaction of the alpha -aminoacrylate with indoline leads to the accumulation of a quinonoid intermediate absorbing at 466 nm (16, 27, 28). When the beta S178P enzyme reacts with indoline, a quinonoid species is formed, and the Kd of indoline is similar (data not shown) to that of the wild type (16).

To correlate structural, regulatory, and functional information, crystals of beta S178P tryptophan synthase were grown. The crystallization conditions were very similar to those previously used to obtain crystals of the wild type (10). The functional and regulatory properties of beta S178P crystals were investigated by polarized absorption microspectrophotometry. Polarized absorption spectra of mutant tryptophan synthase crystals were recorded in the presence of L-serine, with and without sodium ions and with and without IAG (Fig. 7). When L-serine is added to the mutant tryptophan synthase crystals in the absence of cations, the alpha -aminoacrylate is the predominant species, as in the wild type. The presence of sodium ions causes a significant shift of the equilibrium distribution of intermediates, favoring the accumulation of the external aldimine at a higher concentration than in solution (Fig. 5) but lower than in the wild type enzyme (21). Saturating concentrations of IAG cause only a small shift of the equilibrium toward the alpha -aminoacrylate species, indicating that the loss of communication between alpha  and beta  subunits is detectable also in the crystalline enzyme.


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Fig. 7.   Polarized absorption spectra of beta S178P enzyme crystals. Spectra were recorded on crystals suspended in a solution containing 50 mM bis-Tris propane, 20% polyethylene glycol 8000 in the presence of 50 mM L-serine (---), 50 mM L-serine and 250 mM NaCl (--- --- ---), and 50 mM L-serine, 250 mM NaCl, and 10 mM IAG (···), pH 7.8, at 20 °C. Spectra are shown along the direction of polarization where maximal absorption occurred (15).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A model of the structural pathway for the transmission of allosteric signals between alpha  and beta  subunits of tryptophan synthase was proposed on the basis of fluorescence and phosphorescence properties of beta Trp-177 in the absence and presence of alpha  subunit ligands (29, 30). This model was later validated by the crystallographic structures of the enzyme (9, 10, 31-34) and by computational methods (35). Key structural elements of the allosteric communication are loops 2 and 6 of the alpha  subunit and the beta  helix 6 in the COMM domain (9, 10, 33, 34). The beta Ser-178 residue is adjacent to beta Trp-177 and is the last amino acid of the beta  helix 6. Crystallographic studies of 5-fluoroindole-3-propanol phosphate (10) and indole-3-propanol phosphate enzyme complexes (34) showed that the loop 6 of the alpha  subunit interacts with beta  helix 6 via a hydrogen bond between the amidic nitrogen of alpha Gly-181 and the carbonyl oxygen of beta Ser-178. The bond length is 3.4 and 2.8 Å, respectively. A value of 3.9 Å was found in the GP-enzyme complex (9), indicating that bond formation is facilitated by the indole ring of the alpha  subunit ligands interacting with alpha Gly-184 and causing a motion of the alpha  loop 6. In turn, a movement of the entire alpha  loop 2 and, in particular, the displacement of the active site residue alpha Asp-60 are triggered by the relocation of alpha Thr-183. Thus, new polar interactions are formed between alpha  loop 2 and beta  helix 6 with the transmission of signals to the COMM domain (10). A theoretical investigation indicated that motions of the COMM domain are linked to the motions of residues beta 174-179, localized at the entrance of the tunnel (35). Thus, the intramolecular transfer of indole from alpha  to beta  active sites was proposed to be allosterically controlled via the modulation of tunnel accessibility. Recent structural studies of beta A169L/beta C170W mutants have indicated that residue beta 170 is critical in impairing channeling, whereas residue beta 169 is relevant for a correct conformation of the COMM domain (36). The catalytic action of the beta  subunit is allosterically regulated via i) an open-closed transition of alpha  and beta  subunits, ii) a change of conformational flexibility of the beta  subunit, iii) a pH dependence of the equilibrium distribution of intermediates and the catalytic activity, iv) a distinct influence of the monovalent cations sodium, potassium, and cesium, competitively bound to the same site of the beta  subunit, on the activity and distribution of intermediates, and v) effects triggered by binding of alpha  subunit ligands.

The work on the beta S178P mutant was aimed at understanding whether the pathway of communication between alpha  and beta  subunits is unique or whether there are multiple, specialized pathways, each one predominantly involved in controlling a specific dynamic and functional effect on the beta  active site. Modelling and experimental data indicate that the replacement of beta Ser-178 with Pro does not apparently perturb the overall structure of the enzyme and the catalytic activity of the individual subunits within a tight tetrameric complex. Moreover, the alpha  subunit of the mutant enzyme binds ligands with similar affinities as the wild type, indicating that the mutation does not alter the alpha  active site. The major effect of the mutation is the loss of the ability of the alpha  subunit ligands to affect both the beta  subunit activity and the equilibrium distribution of intermediates. The loss of the influence of the alpha  subunit ligands on the beta  subunit is also present in three other mutants of the loop 6 of the alpha  subunit, alpha T183A, Delta alpha 183-185, and alpha R179L (37, 38). alpha Thr-183 interacts with alpha Asp-60 via its hydroxyl residue and seems to be involved in the ordering of the loop 6. alpha Arg-179 forms a series of bonds with other residues of the same loop and is probably involved in the maintenance of a correct position of the loop (10). In contrast, enzymes with mutations in the alpha  loop 2 or in residues contacting the loop do not show any irreversible perturbation of the allosteric regulation of the beta  subunit brought about by alpha  ligands. For example, in the beta K167T mutant the communication between alpha  and beta  subunits is compromised, but the binding of GP can counterbalance this negative effect (26, 39, 40). A strong decrease of allosteric activation was also observed in the beta A169L mutant (36). In the alpha D60N mutant the effect of GP on the equilibrium between the external aldimine and alpha -aminoacrylate is similar to that observed for the wild type enzyme (33). In the alpha D56A and alpha P57A mutants the presence of an alpha  subunit ligand enhances the beta  activity (17, 40). Furthermore, it was shown that the loop 2 interacts only with alpha  subunit ligands that possess an indole ring (9). On the basis of the above considerations, it is possible to propose the pathways of allosteric communication from the alpha  subunit to the beta  subunit shown in Scheme 1.


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Scheme 1.  

When a ligand binds to the alpha  subunit, an interaction takes place with alpha Gly-184 in the loop 6 of the alpha  subunit. The movement of the loop 6 is propagated to alpha Asp-60 via the hydrogen bond with alpha Thr-183. This displacement allows Asp-60 to assume the correct position for catalysis. In agreement with this hypothesis, it was found that alpha T183A and Delta alpha 183-185 mutants are completely inactive (37). Furthermore, the movement of the alpha  loop 6 causes the formation of a hydrogen bond between alpha Gly-181 and beta Ser-178 and a shift of the beta  helix 6 of the COMM domain. The relocation of the beta  helix 6 leads to new interactions between the alpha  loop 2 and the COMM domain, with the formation of polar interactions between alpha Asp-56 and beta Lys-167. Therefore, alpha  loop 6 is critical for the transmission of the allosteric signals originated from alpha  subunit ligands, whereas alpha  loop 2 is critical for intersubunit interactions that increase the activity of both the alpha  and beta  subunits in the tetramer with respect to the isolated subunits.

Another important aspect of the regulatory machinery of tryptophan synthase is the influence of monovalent cations. The binding site for monovalent cations was structurally characterized (9, 10, 31-34). The influence of monovalent cations on the external aldimine/alpha -aminoacrylate distribution was reported (21, 26). Na+ stabilizes the allosterically silent external aldimine species. This cation is lost after indole-3-glycerol phosphate binding, following the COMM domain displacement and the formation of the salt bridge between beta Lys-167 and alpha Asp-56 (31, 34). Furthermore, the dissociation of Na+ appears to determine the movement of two residues, beta Tyr-279 and beta Phe-280, out of the indole tunnel (31). On the other hand, Cs+ binding leads to the accumulation of a tautomeric form of the alpha -aminoacrylate characterized by an absorption peak at 450 nm (21). In the absence of cations, the alpha -aminoacrylate tautomer absorbing at 350 nm is the predominant species (21). In the beta S178P mutant tryptophan synthase the effect of Cs+ is comparable with that on the wild type enzyme. Cs+ binds to the tryptophan synthase with an apparent Kd similar to that of the wild type (21). The behavior of Na+ is very different. The affinity of this cation for the mutant is extremely reduced with respect to the wild type. This might be due to the breaking of the contact between alpha Gly-181 and beta Ser-178, which, in turn, via the movement of beta  helix 6, stabilizes the aminoacrylate conformation, characterized by a low affinity for Na+. The pH dependence of the equilibrium distribution of catalytic intermediates is also altered in the mutant, destabilizing the external aldimine. These findings confirm the relevance of alpha  loop 6 in the control of the equilibrium between active and inactive forms of the enzyme, i.e. between the inactive conformation associated with the external aldimine and the active conformation associated with the alpha -aminoacrylate. In the crystalline state the effect of Na+ is still detectable, probably because the lattice forces prevent the complete displacement of the COMM domain both in the wild type tryptophan synthase and in the mutant enzyme. As a consequence, high concentrations of Na+ influence the equilibrium distribution of intermediates and favor the formation of the external aldimine, although not to the same extent as in the wild type. Nevertheless, the allosteric communication is lost also in the crystalline enzyme, strongly supporting the relevant role of beta Ser-178 in alpha  ligand-triggered intersubunit signals and in the stabilization of the alpha -aminoacrylate conformation. These findings open the way to the structural characterization of an active but allosterically "knocked out" enzyme.

    ACKNOWLEDGEMENT

We are indebted to Dr. Edith Miles, National Institutes of Health, for the gift of the E. coli strain containing the plasmid encoding S. typhimurium tryptophan synthase.

    FOOTNOTES

* This work was supported by funds (to A. M., PRIN99) from the Italian Ministry of University and Scientific and Technological Research and the Italian National Research Council.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.

|| To whom correspondence should be addressed: Inst. of Biochemical Sciences, University of Parma, Parco Area delle Scienze, 43100 Parma, Italy. Tel.: 39-0521-905138; Fax: 39-0521-905151; E-mail: biochim@unipr.it.

Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M011781200

2 A. Marabotti, unpublished observation.

3 An alternative strategy that can be applied, and that we are pursuing, is to mutate alpha Gly-181 to Pro.

4 E. W. Miles, personal communication.

    ABBREVIATIONS

The abbreviations used are: COMM, communication; bis-Tris propane, 1,3-bis[tris(hydroxymethyl)methylamino]propane; MOPS, 3-(N-morpholino)propanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine; PCR, polymerase chain reaction; IAG, indole-3-acetylglycine; GP, D,L-alpha -glycerophosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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