From the 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
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ABSTRACT |
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The
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 2
2 tryptophan synthase complex is a
model enzyme for understanding allosteric regulation. We report the
functional and regulatory properties of the
S178P mutant. Ser-178 is
located at the end of helix 6 of the
subunit, belonging to the
domain involved in intersubunit signaling. The carbonyl group of
Ser-178 is hydrogen bonded to Gly-181 of loop 6 of the
subunit
only when
subunit ligands are bound. An analysis by molecular
modeling of the structural effects caused by the
S178P mutation
suggests that the hydrogen bond involving
Gly-181 is disrupted as a
result of localized structural perturbations. The ratio of
to
subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight
complex. Both the
activity of the
subunit and the inhibitory effect of the
subunit ligands indole-3-acetylglycine and
D,L-
-glycerol-3-phosphate were found to be
the same for the mutant and wild type enzyme, whereas the
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-
-glycerol-3-phosphate do not affect the
activity. Accordingly, the distribution of L-serine
intermediates at the
-site, dominated by the
-aminoacrylate, is only slightly influenced by
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
-aminoacrylate tautomer absorbing at 450 nm.
Crystals of the
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
and
activity in the
2
2 complex with
respect to the isolated subunits results from interactions that involve
residues different from
Ser-178 and (ii)
Ser-178 is a critical
residue in ligand-triggered signals between
and
active sites.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
two
subunits, catalyzing the last two steps in the biosynthesis of
L-tryptophan (1-3) (Fig. 1).
The
active site cleaves indole-3-glycerol phosphate into indole and
D-glyceraldehyde-3-phosphate. The
active site contains
a pyridoxal 5'-phosphate molecule bound to Lys-87 via a Schiff
base and catalyzes the replacement of the
-hydroxyl of
L-serine with indole. The latter compound is
intramolecularly channeled from the
site via a hydrophobic tunnel
connecting the
and
active sites, as proposed on the basis of
the first crystal structure of the enzyme (4). The
and
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
subunit exists either in a
closed, catalytically active state, when the
-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
active site, signals are generated that trigger the activation of the
active site, keeping in phase the action of the two sites. Intersubunit communications are mediated by a movable domain of the
subunit, called the COMM1
(communication) domain, interacting with the mobile loops 2 and 6 of
the
subunit (9, 10). By comparing the three-dimensional structure
of the enzyme in the presence and absence of the
subunit ligand
5-fluoroindole-3-propanol phosphate and the
-aminoacrylate species (10), it was noted that six new polar interactions are formed,
five of which are between residues of
loop 2 and
helix 6, and
only one is between
Gly-181 of loop 6 and
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
and
activity in the
2
2 complex with
respect to the isolated subunits, ii) the reciprocal action of
and
subunit ligands on kinetic, thermodynamic, and dynamic properties
of the opposite active sites, and iii) pH and monovalent cation
modulation of
subunit activity.
View larger version (88K):
[in a new window]
Fig. 1.
Top panel, three-dimensional structure
of the /
dimer of the tryptophan synthase from the file 1a50.pdb
(10). The
subunit is on the left (light gray), and the
subunit is on the right (dark gray). The coenzyme
pyridoxal 5'-phosphate and the
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
and
reactions of the tryptophan synthase.
To gain insight into the role of Ser-178-
Gly-181 interaction in
regulating the allosteric communication,
Ser-178 was mutated to a proline residue. The rationale for the choice of proline was that the interaction between
Ser-178 and
Gly-181 is mediated by a hydrogen bond between the carbonyl oxygen of
Ser-178 and the
amidic nitrogen of
Gly-181.
Ser-178 is not a conserved residue, as demonstrated by a multiple alignment of the amino acid
sequence of
subunits from different
sources.2 Therefore,
proline mutation was chosen for its unique feature of preventing the
formation of the hydrogen bond with
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.
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MATERIALS AND METHODS |
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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.
and
Activity Assays--
The
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
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 -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
/
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 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
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 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
and
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 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
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.
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RESULTS |
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Because the three-dimensional structure of the 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
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
Ser-178 and
Gly-181. No other significant movements appear to take place either in the
or
subunit. An experimental evaluation of the effect of mutation on the strength of
the
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
and
subunit concentrations is 0.7, as for the wild type (17), indicating that the
mutation does not appreciably affect the intersubunit affinity of the
2
2 mutant complex.
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The subunit activity of the mutant was found to be very similar to
that of the wild type, and the
subunit activity was about 2-fold
lower. Furthermore, the
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
active site geometry and does not impair the binding of
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
subunit activity was detected (Fig. 3,
c and d), indicating that this feature of the allosteric regulation between
and
subunits is lost in the
S178P mutant.
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A well characterized effect of the allosteric regulation is
the change of the equilibrium distribution of catalytic intermediates at the
active site caused by
subunit ligands (2, 5, 8, 15, 16,
20). This distribution is also affected by pH, monovalent cations, and
temperature (8, 21). Specifically,
subunit ligands, low pH, cesium
ions, and high temperature stabilize the
-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
regulation, the
influence of
subunit ligands, monovalent cations, and pH on the
equilibrium distribution of catalytic intermediates of the
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
-aminoacrylate, mainly absorbing at 350 nm (Fig. 4b), as observed for the wild type (21). When a
solution of the
-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
-aminoacrylate absorbing at 450-470 nm,
as observed in O-acetylserine sulfhydrylase (22) and
cystathionine
-synthase (23, 24). This result indicates a negligible
influence of IAG and GP on the equilibrium distribution of
-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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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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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
-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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In the wild type enzyme the reaction of the -aminoacrylate with
indoline leads to the accumulation of a quinonoid intermediate absorbing at 466 nm (16, 27, 28). When the
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 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
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
-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
-aminoacrylate species, indicating that the
loss of communication between
and
subunits is detectable also
in the crystalline enzyme.
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DISCUSSION |
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A model of the structural pathway for the transmission of
allosteric signals between and
subunits of tryptophan synthase was proposed on the basis of fluorescence and phosphorescence properties of
Trp-177 in the absence and presence of
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
subunit and the
helix 6 in the COMM domain (9, 10, 33, 34). The
Ser-178 residue is adjacent to
Trp-177 and is the last amino acid of the
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
subunit interacts with
helix 6 via a hydrogen bond between the amidic nitrogen of
Gly-181 and the
carbonyl oxygen of
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
subunit ligands interacting with
Gly-184 and causing a motion of
the
loop 6. In turn, a movement of the entire
loop 2 and, in
particular, the displacement of the active site residue
Asp-60 are
triggered by the relocation of
Thr-183. Thus, new polar interactions
are formed between
loop 2 and
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
174-179, localized at the entrance of the tunnel (35). Thus, the
intramolecular transfer of indole from
to
active sites was
proposed to be allosterically controlled via the modulation of tunnel
accessibility. Recent structural studies of
A169L/
C170W mutants
have indicated that residue
170 is critical in impairing channeling,
whereas residue
169 is relevant for a correct conformation of the
COMM domain (36). The catalytic action of the
subunit is
allosterically regulated via i) an open-closed transition of
and
subunits, ii) a change of conformational flexibility of the
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
subunit, on the activity and
distribution of intermediates, and v) effects triggered by binding of
subunit ligands.
The work on the S178P mutant was aimed at understanding whether the
pathway of communication between
and
subunits is unique or
whether there are multiple, specialized pathways, each one
predominantly involved in controlling a specific dynamic and functional
effect on the
active site. Modelling and experimental data indicate
that the replacement of
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
subunit of the mutant enzyme binds ligands with similar affinities
as the wild type, indicating that the mutation does not alter the
active site. The major effect of the mutation is the loss of the
ability of the
subunit ligands to affect both the
subunit
activity and the equilibrium distribution of intermediates. The loss of
the influence of the
subunit ligands on the
subunit is also
present in three other mutants of the loop 6 of the
subunit,
T183A,
183-185, and
R179L (37, 38).
Thr-183 interacts
with
Asp-60 via its hydroxyl residue and seems to be involved in the
ordering of the loop 6.
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
loop 2 or in residues contacting the loop do not
show any irreversible perturbation of the allosteric regulation of the
subunit brought about by
ligands. For example, in the
K167T
mutant the communication between
and
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
A169L mutant (36). In the
D60N mutant the effect of GP on the
equilibrium between the external aldimine and
-aminoacrylate is
similar to that observed for the wild type enzyme (33). In the
D56A
and
P57A mutants the presence of an
subunit ligand enhances the
activity (17, 40). Furthermore, it was shown that the loop 2 interacts only with
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
subunit
to the
subunit shown in Scheme
1.
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When a ligand binds to the subunit, an interaction takes place with
Gly-184 in the loop 6 of the
subunit. The movement of the loop 6 is propagated to
Asp-60 via the hydrogen bond with
Thr-183. This
displacement allows Asp-60 to assume the correct position for
catalysis. In agreement with this hypothesis, it was found that
T183A and
183-185 mutants are completely inactive (37).
Furthermore, the movement of the
loop 6 causes the formation of a
hydrogen bond between
Gly-181 and
Ser-178 and a shift of the
helix 6 of the COMM domain. The relocation of the
helix 6 leads to
new interactions between the
loop 2 and the COMM domain, with the
formation of polar interactions between
Asp-56 and
Lys-167.
Therefore,
loop 6 is critical for the transmission of the
allosteric signals originated from
subunit ligands, whereas
loop 2 is critical for intersubunit interactions that increase the
activity of both the
and
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/-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
Lys-167 and
Asp-56 (31, 34). Furthermore, the dissociation of Na+ appears to determine the movement
of two residues,
Tyr-279 and
Phe-280, out of the indole tunnel
(31). On the other hand, Cs+ binding leads to the
accumulation of a tautomeric form of the
-aminoacrylate
characterized by an absorption peak at 450 nm (21). In the absence of
cations, the
-aminoacrylate tautomer absorbing at 350 nm is the
predominant species (21). In the
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
Gly-181 and
Ser-178, which, in turn, via the movement of
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
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
-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
Ser-178 in
ligand-triggered intersubunit signals and in the
stabilization of the
-aminoacrylate conformation. These findings
open the way to the structural characterization of an active but
allosterically "knocked out" enzyme.
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ACKNOWLEDGEMENT |
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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.
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FOOTNOTES |
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* 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 Gly-181 to Pro.
4 E. W. Miles, personal communication.
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ABBREVIATIONS |
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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--glycerophosphate.
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