(Received for publication, March 10, 1995; and in revised form, May 15, 1995)
From the
We are using the tryptophan synthase
The regulation of enzyme activity and of substrate specificity
is a problem of critical importance in biology. Enzyme activity can be
modulated by interaction of the enzyme with ligands or with other
proteins. These interactions produce changes in protein conformation or
alter the equilibrium distribution of preexisting conformations.
Mutations can also modulate enzyme activity by altering protein
conformation. In the present work we ask whether ligands that stabilize
the active conformation of an enzyme can also repair the deleterious
effects of a mutation that leads to an inactive conformation. Tryptophan synthase (EC 4.2.1.20) is a classic system for
investigating how ligands, protein-protein interaction, and mutations
regulate enzyme activity and substrate specificity (for reviews, see (1, 2, 3, 4, 5) ). The
bacterial enzyme is an Investigations of the reaction and substrate specificities of wild
type and mutant forms of the
Association of the Early studies showed that some
monovalent cations (K Because certain cations and
Figure 1:
Effects of CsCl concentration on
activity in the
The results reported here were all carried out in 50 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA at pH 7.8, since we had used this buffer for previous related
experiments(17) . Although Na
Fig. 1shows the effects of CsCl
concentration on activity in the The activity
data in Fig. 1can be represented by a model (see Table 3)
which assumes the noncooperative binding of CsCl to one or two classes
of sites on the protein with average dissociation constants K
Figure 2:
Effects of CsCl concentration on
absorption spectra in the presence or absence of 50 mML-serine. A, wild type
Figure SI:
Scheme I. Reactions of the wild type
tryptophan synthase
Globular proteins are flexible molecules and can have two or
more conformations in solution. Ligand or substrate binding may produce
changes in protein conformation or alter the equilibrium distribution
of preexisting conformations. The results reported above show the
effects of two ligands (Cs
Related previous studies
have shown that addition of a high concentration of
NH
The crystal structure of pyridoxal
phosphate-dependent 2,2-dialkylglycine decarboxylase reveals the
location of two binding sites for alkali metal ions. One is located
near the active site and accounts for the dependence of activity on
K
In conclusion, to explain our
finding that the wild type
complex as a model system to
investigate how ligands, protein-protein interaction, and mutations
regulate enzyme activity, reaction specificity, and substrate
specificity. The rate of conversion of L-serine and indole to L-tryptophan by the
subunit alone is quite
low, but is activated by certain monovalent cations or by association
with
subunit to form an
complex. Since monovalent cations and
subunit appear to
stabilize an active conformation of the
subunit, we
have investigated the effects of monovalent cations on the activities
and spectroscopic properties of a mutant form of
complex having
subunit glutamic acid 109 replaced by alanine (E109A). The E109A
complex is inactive in reactions
with L-serine but active in reactions with
-chloro-L-alanine. Parallel experiments show effects of
monovalent cations on the properties of wild type
subunit and
complex. We find
that CsCl stimulates the activity of the E109A
complex and of wild type
subunit with L-serine and indole and alters the
equilibrium distribution of L-serine reaction intermediates.
The results indicate that CsCl partially repairs the deleterious
effects of the E109A mutation on the activity of the
complex by stabilizing a
conformation with catalytic properties more similar to those of the
wild type
complex. This conclusion
is consistent with observations that monovalent cations alter the
catalytic and spectroscopic properties of several pyridoxal
phosphate-dependent enzymes by stabilizing alternative conformations.
complex that
dissociates reversibly into monomeric
subunits and dimeric
subunits. The separate
subunit catalyzes the
reversible aldolytic cleavage of indole-3-glycerol phosphate to D-glyceraldehyde 3-phosphate and indole, termed the
reaction. The separate
subunit catalyzes the
condensation of L-serine with indole to form L-tryptophan in a pyridoxal phosphate-dependent
-replacement reaction, termed the
reaction. The interaction
of the
and
subunits in the
complex serves to increase the rates
of the
and
reactions up to 100-fold, increase substrate
binding affinities, and alter reaction specificities. These changes are
attributed to conformational changes which occur upon assembly of the
complex(6) . The binding of a ligand or the formation of a
reaction intermediate at the active site of either subunit in the
complex alters the reaction kinetics
at the heterologous active site
25 Å
distant(7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
The combined results suggest that the
and
subunits undergo conformational changes during catalysis and
exist in two or more conformations that have been designated
``open'' and ``closed'' (11) .
subunit and the
complex also provide evidence that
the
subunit can exist in two different conformational
states (designated I and II in Table 1)(17, 18) . The separate
subunit catalyzes
-replacement and
-elimination
reactions with a variety of amino acids that have an electronegative
substituent in the
-position (e.g.L-serine and
-chloro-L-alanine)(19, 20) .
subunit with the
subunit greatly increases activity in the
-replacement reaction
with L-serine and indole and almost completely eliminates
activity in the
-elimination reaction with L-serine. Thus
association with the
subunit alters the reaction specificity of
the
subunit. Whereas L-serine is a better
substrate than
-chloro-L-alanine for the
-replacement reaction with indole catalyzed by the
complex, the reverse is true for the
subunit(17) . Thus association with the
subunit alters the substrate specificity of the
subunit. Several
complexes
with single amino acid replacements in the active site of the
subunit have substrate and reaction specificities
similar to those of the wild type
subunit(17) . This finding led us to suggest that the
wild type
subunit and these mutant
complexes exist in open
conformations (I in Table 1), whereas the wild type
complex exists in a closed
conformation (II in Table 1)(17) . Aliphatic alcohols
also stabilize an alternative conformation of the
complex that has properties similar
to that of the free
subunit in aqueous solution (21) (I in Table 1).
, Li
, and
NH
, but not Na
) activate
the
subunit (22, 23) in
-replacement and
-elimination reactions with L-serine. High concentrations of NH
in some ways mimic the effects of the
subunit on the
kinetic and spectroscopic properties of the
subunit(24, 25, 26) . Recently it has
been shown that monovalent cations (Na
,
K
, Li
, Rb
,
NH
, and Cs
) activate the
wild type tryptophan synthase
complex and alter the pH-dependent equilibrium distribution of
enzyme-substrate
intermediates(27, 28, 29, 30, 31) .
Together the results support the idea that both the free
subunit and the
complex exist
in two or more conformations. Preferential binding of certain
monovalent cations to a specific site in the
subunit
may alter the equilibrium distribution of preexisting conformations in
the
subunit and
complex by stabilizing conformation II (Table 1). Binding
of an
subunit ligand (
-glycerol 3-phosphate) to the
complex also alters the distribution
of intermediates formed with L-serine(32) . Table 1indicates that
site ligands stabilize conformation
II.
site ligands appear to
stabilize the more active conformation II of wild type
subunit and
complex (Table 1), we reasoned that these ligands might promote the
conversion of a mutant
complex from
an inactive conformation I to an active conformation II. In the present
work we have investigated the effects of monovalent cations on a mutant
complex having Glu-109 of the
subunit replaced by alanine (E109A). Glu-109 is
located in a region of the
subunit adjacent to the
covalently bound pyridoxal phosphate cofactor and near the putative
indole-binding site of the
subunit (see Fig. 1in (17) ). It has been postulated that the
carboxylate of Glu-109 may activate the indole toward nucleophilic
attack on the aminoacrylate (33) and/or activation of the
-hydroxyl of L-serine as a leaving group(18) .
The finding that the E109A
complex
can synthesize L-tryptophan from
-chloro-L-alanine and indole but not from L-serine and indole provides evidence that Glu-109 is not an
essential residue for the activation of indole but may serve to
activate the
hydroxyl of L-serine as a leaving
group(17, 18, 34) . Alternatively,
replacement of Glu-109 by alanine (17) or aspartate (16, 18) may introduce structural changes within the
subunit that alter the conformation of the
complex and thereby alter the
substrate and reaction specificity. Our finding that the E109A
complex resembles the wild type
subunit in substrate specificity and reaction
specificity suggests that the mutation stabilizes conformation I (see
``mutations'' in Table 1). The results reported here
show that addition of a high concentration of CsCl partially restores
the activity of the E109A
complex
with L-serine and alters the substrate and reaction
specificity. The results suggest that CsCl repairs a conformational
defect in the E109A
complex by
stabilizing conformation II that has structural and catalytic
properties more similar to those of the wild type
complex.
-replacement reaction with L-serine and
indole. A, wild type
complex - GP (
) or + 10 mM GP
(
); B, wild type
subunit (
); C, E109A
complex - GP
(
) or + 10 mM GP
(
).
Chemicals and Buffers
DL--Glycerol
3-phosphate (GP),
(
)
-chloro-L-alanine (hydrochloride), and
pyridoxal phosphate were from Sigma. All spectroscopic experiments and
enzyme assays utilized Buffer B (50 mM sodium N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA
at pH 7.8).
Enzymes
Wild type (35) and E109A (36) forms of the tryptophan synthase
complex and wild type
subunits (37) from Salmonella typhimurium were
isolated and purified to homogeneity. Protein concentrations were
determined from the specific absorbance at 278 nm of the
complex (E
= 6.0) or of the
subunit (E
= 6.5)(38) .
Enzyme Assays
One unit of activity in any reaction
is the formation of 0.1 µmol of product in 20 min at 37 °C.
-Replacement reactions with L-serine or
-chloro-L-alanine and indole were measured by a direct
spectrophotometric assay (38) containing modified components
(100 mML-serine or 40 mM
-chloro-L-alanine, respectively, and 0.2 mM indole in Buffer B).
Spectroscopic and Analytical Methods
Absorption
spectra were made using a Hewlett-Packard 8452 diode array
spectrophotometer. Time course measurements of enzymatic activities at
single wavelengths were made using a Cary 118 spectrophotometer.
does not
activate the separate
subunit(22, 23) , recent studies show that
Na
does activate the wild type
complex(27, 28, 29, 30, 31) .
These recent studies utilized triethanolamine and bis-tris propane
buffers that contain no monovalent cations and are thus better suited
for studies of the effects of cations. Because Na
in
our buffer (
50 mM) may compete with other cations for the
same binding site, our results do not show the effects of the absolute
concentrations of other cations. However, our results do show that
other cations have striking effects on the properties of the wild type
subunit and
complex and on the E109A
complex.
Effects of Monovalent Cations on Activity
Table 2shows the effects of various monovalent cations as
their chlorides (0.2 M) on the specific activity of the wild
type subunit in the
-replacement reactions with L-serine and indole and with
-chloro-L-alanine
and indole. Addition of Li
or
NH
significantly stimulates activity with L-serine and indole, as found previously with partially
purified
subunit from Escherichia
coli(23) . Stimulation of the
subunit by
Rb
and Cs
is reported for the first
time. Because Cs
gave the greatest stimulation,
Cs
was selected for further study. The monovalent
cations have much smaller effects on the activities with
-chloro-L-alanine plus indole. Thus the monovalent
cations that increase the activity with L-serine and indole
alter the substrate specificity by increasing the ratio of activity
with L-serine to activity with
-chloro-L-alanine
(see serine:
-chloro-L-alanine activity ratio in Table 2). Note that the ratio of activities with L-serine and
-chloro-L-alanine is one of the
features that distinguish the postulated conformations I and II (Table 1).
-replacement reaction with L-serine and indole. Low concentrations of CsCl activate the
wild type
complex (A) and
subunit (B). High concentrations of CsCl
activate the E109A
complex (C) but inhibit the wild type
complex (A). The
subunit ligand, GP, alters the
effect of CsCl concentration on the activities of the wild type and
E109A
complexes.
and K
. The binding of CsCl
converts the enzyme from the CsCl free form X to CsCl-bound forms Y or
Z, with concomitant changes in activity. Addition of higher
concentrations of CsCl (100-3000 mM) to the wild type
complex (Fig. 1A)
produces further changes of the activity which vary monotonically with
salt concentration. These changes could result from additional binding
at very weak sites on the protein or from the non-ideal behavior of
concentrated salt solutions. For the purpose of curve fitting, these
changes are expressed as a linear function of salt concentration and
described as a ``solvent effect'' in Table 3. The
curves shown in Fig. 1, A-C, were the best fits
to this model. Table 3describes the model and the derived
dissociation constants. The results give no indication of the
stoichiometry of the reaction between CsCl and the enzyme.
Effects of CsCl on Absorption Spectra
The
absorption spectra of the wild type complex (Fig. 2A),
subunit (Fig. 2B), and E109A
complex (Fig. 2C) are very similar in the absence
of L-serine. Each enzyme exhibits a major peak centered at 412
nm due to the internal aldimine formed between pyridoxal phosphate and
subunit Lys-87 (E in Fig. SI).
Reaction of the wild type
complex
with L-serine in the presence or absence of CsCl yields a
complex spectrum with a major peak centered at 340 nm (Fig. 2A), which is ascribed to the pyridoxal
phosphate-amino acrylate Schiff base (E-AA in Fig. SI).
Reaction of the
subunit (Fig. 2B) or
E109A
complex (Fig. 2C) with L-serine in the absence of CsCl
yields an absorption spectrum with maximum absorbance at 424 nm. This
intermediate is the external aldimine of pyridoxal phosphate with L-serine (E-Ser in Fig. SI) and exhibits an
intense fluorescence emission at 510 nm. Addition of increasing
concentrations of CsCl to the
subunit (Fig. 2B) or the E109A
complex (Fig. 2C) results in decreased absorbance
at 424 nm, increased absorbance at 340 nm, and decreased fluorescence
emission at 510 nm (fluorescence data not shown). Thus CsCl alters the
equilibrium distribution of ES-Ser and ES-AA
intermediates with the
subunit and the E109A
complex.
complex; B, wild type
subunit; C, E109A
complex.
complex.
and
-glycerol
3-phosphate) on the catalytic properties and spectroscopic properties
of wild type and mutant forms of tryptophan synthase. We conclude that
Cs
stabilizes the active conformation of tryptophan
synthase and partially repairs the deleterious effects of a mutation
that leads to an inactive conformation.
Cs
Our analysis (Table 3) of the
activity data in Fig. 1indicates that CsAlters the Catalytic Properties of
Tryptophan Synthase
binds
to two classes of sites in the wild type and E109A
complexes and to one class of sites
in the wild type
subunit. Low concentrations of
Cs
activate the wild type
complex (K
= 25 mM)
and the wild type
subunit (K
= 43 mM). Because
50 mM Na
is present in our assay mixture, the activity
of the wild type
complex in the
absence of CsCl is significantly higher than the activity in the
absence of any monovalent
cation(27, 28, 29, 30, 31) .
The cited results indicate that monovalent cations convert the wild
type
complex from conformation I
that is less active in the
reaction to a more active conformation
II as shown in Table 1. Our results in Table 3indicate
that low concentrations of Cs
convert the wild type
complex from a less active form X to
a more active form Y. High concentrations of Cs
decrease the activity of the wild type
complex. The shape of the curve in Fig. 1A (-GP) suggests that these high
concentrations of CsCl may have a solvent effect and favor a less
active conformation Z (Table 3). Since 3 M CsCl does not
alter the far- and near-UV circular dichroism spectra of the wild type
complex (data not shown), effects of
this high concentration of CsCl must have subtle, rather than gross,
effects on the conformation. Addition of GP, an analogue of the
subunit substrates indole-3-glycerol phosphate and D-glyceraldehyde 3-phosphate, inhibits the activity of the
wild type
complex in the
reaction (Fig. 1A) as found previously(39) .
Inhibition is greatest near the concentration of CsCl that gives
maximum activity in the absence of GP. This result suggests that the
most active form of the
complex
(form Y in Table 3) is most susceptible to this allosteric
inhibition. It is possible that the step which is inhibited by
-glycerol 3-phosphate is rate-limiting for form Y of the
complex but is only partially
rate-limiting for forms X and Z.
Cs
Our finding CsCl
alters the equilibrium distribution of enzyme-substrate intermediates (ES-Ser and ES-AA in Fig. SI) with the
Alters the Spectroscopic
Properties of the
Subunit
subunit (Fig. 1B) is consistent with
early studies on the effects of NH
on the
spectroscopic properties of the
subunit with L-serine(24) . NH
was
also found to alter the presteady state kinetics of interconversion of ES-Ser and ES-AA(26) . In some ways high
concentrations of NH
mimic the effects of
the
subunit on the properties of the
subunit.
NH
or
subunit increases the rate of
formation of E-Ser and increases the rate of conversion of E-Ser to E-AA (Fig. SI). The conversion of E-Ser to E-AA is rate-limiting in the absence of
NH
or
subunit(25) .
Cs
Addition of
CsPartially Repairs a
Conformational Defect in a Mutant Enzyme
to the
complex
having the E109A mutation in the
subunit partially
restores the activity with L-serine and indole (Fig. 1C) and makes the spectroscopic properties in the
presence of L-serine (Fig. 2C) more like those
of the wild type
complex (Fig. 2A). These results provide evidence that a high
concentration of Cs
stabilizes conformation II of the
E109A
complex that is more active in
the
reaction. Alternatively, high concentrations of Cs
might prevent dissociation of the E109A
complex to separate
and
subunits under assay conditions. A strong argument
against this possibility is provided by our previous finding that
addition of GP results in an 80% inhibition of the activity of the wild
type and E109A
complexes in the
-replacement reaction with
-chloro-L-alanine and
indole, but does not inhibit the corresponding activity of the separate
subunit(17) .
partially restores the activity of a
mutationally altered form of the
subunit found in E. coli strain A2B17(22) . This type of mutant has
been termed ``repairable'' because it has activity with L-serine in the
reaction in the presence, but not in the
absence, of the
subunit. Ammonium ions often work synergistically
with
subunits in the activation of other repairable
subunits(40) . A high concentration of
NH
also partially restores the activity of
a mutant form of the
subunit (B8) that has an amino
acid substitution in the ``hinge''
region(41, 42) . The results suggest a functional role
for the hinge region in the process of conformational switching.
Studies in progress in our laboratory reveal that moderate
concentrations of Cs
or NH
also partially restore the activities of a number of other mutant
forms of the
subunit and
complex. Taken together the results of these studies support the
model in Table 1that monovalent cations and the
subunit
stabilize conformation II of the wild type and mutant
subunits that is more active in the
reaction.
Crystallographic Studies of Cation Binding Sites in
Pyridoxal Phosphate Enzymes
It is important to obtain additional
evidence for the nature of the conformational change postulated in Table 1. Three other pyridoxal phosphate-dependent enzymes which
are activated by monovalent cations (tyrosine
phenol-lyase(43, 44) ,
tryptophanase(44, 45, 46) , and
dialkylglycine decarboxylase(47) ) have recently been examined
by x-ray
crystallography(48, 49, 50, 51, 52) .
(For an excellent recent review, see (30) ). Because the
available x-ray structures show that the monovalent cation binding
sites are not located in the active site, the bound cations should be
classified as allosteric effectors(30) . These allosteric
effectors may activate the enzymes by stabilizing the active
conformation or may play a more dynamic role. For an enzyme such as
tryptophan synthase that catalyzes a reaction through a series of steps
(see Fig. SI) monovalent cation binding could selectively lower
the activation energy for a particular step and thus alter the
equilibrium distribution of intermediates and the kinetics of specific
steps(30) . or
Rb
(49, 51, 52, 53) .
The exchange of Na
for K
at the
location near the active site results in a gross change in the
coordination geometry, in concerted rearrangement of the conformations
of Ser
and Tyr
, and in small structural
changes extending far beyond the immediate surroundings of the metal
ion(49, 51, 52) . The changes in the
conformations of Ser
and Tyr
, which are in
the active site, may disrupt the productive binding of substrate. This
change in conformation could drive the enzyme into an inactive (or low
activity form) in which the bound substrate and the active site
functional groups and/or the pyridoxal phosphate ring are improperly
aligned for catalysis(30) . These important studies provide the
groundwork for understanding the structural and functional roles of
monvalent cations in other pyridoxal phosphate enzymes.
Structure and Function of Tryptophan Synthase
The
three-dimensional structure of the tryptophan synthase
complex from S. typhimurium has been reported (53) and described in several
reviews(3, 5, 30) . Because the active sites
of the
and
subunits are
25 Å apart,
the observed effects of ligands that bind to each subunit on the
properties of the partner subunit are allosteric in nature. Thus
ligands that bind to the active site of the
subunit produce
allosteric effects on the individual steps in reactions catalyzed by
the
subunit (Fig. SI). Studies by Peracchi et al.(28, 29) and Dunn (27, 30, 31) and our studies reported herein
show that monovalent cations also act as allosteric effectors of these
subunit reactions. In addition, Woehl and Dunn (30, 31) find that monovalent cations are essential
for the allosteric activation of the
site by formation of E-AA (see Fig. SI) at the
site. The cited results
and results herein support a role for monovalent cations as a switch
that converts the low activity conformation I to a high activity
conformation II (Table 1). Crystallographic studies of the
tryptophan synthase
complex in
progress show that Na
, K
, or
Cs
binds to a site near, but not in, the active site
of the
subunit.
(
)The results
of these studies should give new insights into the roles of monovalent
cations in tryptophan synthase.
subunit of tryptophan
synthase and a mutant form of the
complex (
subunit-E109A) have no or low activity
in reactions with L-serine but high activity in reactions with
-chloro-L-alanine, we propose that that these enzymes
exist in a conformation (I) that results in the improper alignment of
the weak hydroxyl leaving group of L-serine for
-elimination. We suggest that bound Cs
stabilizes
conformation II in which the hydroxyl group of L-serine is
properly aligned for
-elimination. Thus Cs
may
stabilize alternative conformations of the wild type
subunit and E109A
complex that
have structural and catalytic properties more similar to those of the
wild type
complex.
and Carbonyl
Catalysis and the Third Symposium on PQQ and Quinoproteins in Capri,
Italy, May 22-27, 1994. The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
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-glycerol 3-phosphate; bis-tris,
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
and PQQ (Marino, G., Sannia, G., and Bossa, F., eds) pp. 119-124, Birkhauser Verlag, Basel/Switzerland
and PQQ (Marino, G., Sannia, G., and Bossa, F., eds) pp. 125-129, Birkhauser Verlag, Basel/Switzerland
and PQQ (Marino, G., Sannia, G., and Bossa, F., eds) pp. 183-185, Birkhauser Verlag, Basel/Switzerland
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