(Received for publication, October 30, 1996, and in revised form, January 9, 1997)
From the Enzyme Structure and Function Section, To assess the functional roles of helix 13 and of
the conserved and variable residues in the C-terminal region (residues
378-397) of the tryptophan synthase The bacterial tryptophan synthase
Alignment of the sequences of the tryptophan synthase
To investigate the roles of helix 13 and of the conserved and variable
residues in the C-terminal region, we have constructed four C-terminal
truncations and 12 point mutations (Fig. 2) using methods based on
PCR1 (6, 9). The effects of these mutations
on kinetic and spectroscopic properties and thermal stability are
reported here. Our results show that residues in the C-terminal region
are important for the thermal stability and activity of the
Enzymes and chemicals used for
recombinant DNA methods and DNA sequencing were as before (6).
Indole-3-glycerol phosphate was prepared as described (17).
The Escherichia coli host strain CB149
lacks the trp operon (17). Plasmids pEBA-10, pEBA-6, and pEBA-4A8 (6)
express the S. typhimurium tryptophan synthase
The The expression vectors pEBA-10 and
pEBA-6 were used as templates for quick and convenient mutagenesis by
megaprimer PCR (6). Mutagenic primers used in the construction of the
missense mutations and C-terminal truncations were as follows, where
base changes are underlined and mixed bases at the same position are in
parentheses: R379P, R379K, or R379Q, 5 One unit of activity is the amount of enzyme
that gives rise to formation of 0.1 µmol of product per 20 min at
37 °C. Activities of the Absorption spectra utilized a
Hewlett-Packard 8452 diode array spectrophotometer. Fluorescence
measurements were made in a
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
subunit, we have constructed
four C-terminal truncations and 12 point mutations. The effects of these mutations on kinetic and spectroscopic properties and thermal stability are reported here. The mutant
subunits all form stable
2
2 complexes that have been
purified to homogeneity. The mutant
2
2
complexes are divided into two classes on the basis of activity in the
reaction of L-serine with indole to form tryptophan. Class I enzymes, which have mutations at Arg-379 or Asp-381 or truncations (384-397 or 385-397), exhibit significant activity (1-38% of wild type). Class II enzymes, which have mutations at Lys-382 or Asp-383 or
truncations (382-397 or 383-397), exhibit very low activity (<1% of
wild type). Although Class II enzymes have drastically reduced activity
in the reaction of L-serine with indole and an altered
distribution of enzyme-substrate intermediates in the reaction of
L-serine with
-mercaptoethanol, they retain activity in
the reaction of
-chloro-L-alanine with indole.
Correlation of the results with the three-dimensional structure of the
2
2 complex suggests that Lys-382 and
Asp-383 serve important roles in a proposed "open" to "closed"
conformational change that occurs in the reactions of
L-serine. Because mutant
subunits having C-terminal
truncations (383-397 or 384-397) undergo much more rapid thermal
inactivation at 60 °C than the wild type
subunit, the C-terminal
helix 13 stabilizes the
subunit.
2
2
complex (EC 4.2.1.20) is a useful model system for probing the
relationships between enzyme structure and function and the mechanisms
of intersubunit communication (for reviews see Refs. 1-4). The
three-dimensional structure of the
2
2
complex from Salmonella typhimurium revealed that the four
polypeptide chains are arranged in a nearly linear
order
(5). The larger pyridoxal phosphate-dependent
subunit
contains two domains of nearly equal size, termed the N-domain
(residues 1-52 and 85-204 shown in yellow in
Fig. 1A) and the C-domain (residues 53-84
and 205-397 shown in cyan and red in Fig.
1A). The pyridoxal phosphate coenzyme is located in the
interface between these two domains and interacts with residues from
both domains. Portions of the two domains possess a high level of
structural homology and are nearly superimposable, suggesting that they
may have evolved by gene duplication and fusion. The core region of the
C-domain terminates at residue 377 and thus excludes the C-terminal
residues 378-397. Residues 383-393 form a helix (helix 13) that
protrudes from the center of the
subunit into solvent. Three
residues in helix 13 (Ile-384, His-388, and Leu-391) are involved in
/
interaction (Fig. 1B). The side chains of Lys-382
and Glu-350 form a salt bridge located below the plane of the
pyridoxal phosphate ring in the active center of the
subunit
(Fig. 1B and Fig. 5 in Ref. 6).
Fig. 1.
A, ribbon diagram of the
three-dimensional structure of the tryptophan synthase
2
2 complex from S. typhimurium
(5). The
subunits are in green, the N-domain of the
subunit (residues 1-52 and 85-204) is in yellow; the
C-domain residues 53-84 and 205-382 are in cyan, and
residues 378-393 are in red; C-terminal residues 394-397
are not visible in the structure and are not shown. The pyridoxal
phosphate coenzyme is in black. B, stereo ribbon
diagram of the central section from A. Structural elements are labeled according to Ref. 5:
,
-helix;
,
strand. Side chains that are deleted or mutated in this work (see Fig. 2) are shown
in lavender. Side chains of Glu-350 and Gln-142 are shown in
green.
[View Larger Version of this Image (61K GIF file)]
subunit or
domain from 24 species (see supplementary material in the
electronic appendix to Ref. 7) reveals that residues corresponding to
384-397 of the
subunit from S. typhimurium are
variable, whereas the preceding residues 378-383 are identical
(Fig. 2). Several other pyridoxal phosphatedependent
enzymes, including dehydratases and synthases, have also been aligned
with the tryptophan synthase
subunit and assigned to the
family
(8) or to Fold type II (7). The sequence similarity of these other
enzymes with the
subunit is very low in the C-terminal region
(beyond Glu-350 in the
subunit from S. typhimurium).
Fig. 2.
Sequence of C-terminal residues 378-397 of
the tryptophan synthase subunit from S. typhimurium and
structural elements found in the three-dimensional structure (5)
(r, random coil; h, helix; ?,
cannot be seen). Multiple alignment of 24
subunit or domain
sequences (7) shows that residues corresponding to 378-383
(underlined) of the
subunit from S. typhimurium are identical, whereas 384-397 are variable. We have
constructed and characterized the four indicated C-terminal truncation
mutant proteins and 12 mutant proteins having single amino acid
replacements at positions 379, 381, 382, and 383 marked by *; the
construction and initial characterization of five mutant
subunits
altered at position 382 has been reported recently (6).
[View Larger Version of this Image (14K GIF file)]
2
2 complex but are not essential for
catalysis. Alteration or deletion of Lys-382 or Asp-383 drastically
reduces activity in the reaction of L-serine with indole
and alters the substrate specificity and spectroscopic properties. We
correlate these results with data on the three-dimensional structure of
the
2
2 complex (5,
10)2 and with models depicting
ligand-mediated conformational changes of the tryptophan synthase
2
2 complex (11-16). Our results suggest that Lys-382 and Asp-383 are involved in an open to closed
conformational transition that activates the
2
2 complex. Initial aspects of portions
of this work have been reported (6, 9).
Enzymes and Chemicals
-Chloro-L-alanine,
D-glyceraldehyde-3-phosphate dehydrogenase, and other
common chemicals were from Sigma. Buffer B was 50 mM sodium
N,N-bis(2-hydroxyethyl)glycine containing 1 mM EDTA at pH 7.8. A synthetic peptide
(NH2-DIFTVHDILKA-COOH), corresponding to
subunit
residues 383-393, was custom synthesized by Bio-Synthesis Corp.,
Lewisville, TX.
2
2 complex,
subunit, and
subunit,
respectively. Cultures of the host harboring wild type or mutant
plasmids (see below) were grown, and enzymes were induced with IPTG as
described (6). Enzymes were purified from the disrupted cells from
100-ml cultures. Purification of the wild type and mutant
2
2 complexes (18) utilized
crystallization from crude extracts followed by recrystallization. The
combined yields of
and
subunits were greater than 70% of the
total soluble proteins, as reported recently for the five complexes with mutations in
subunit residue 382 expressed under the same conditions (6). The amounts of purified
2
2 complex obtained from 100-ml cultures
ranged from 23 to 90 mg. Each purified
2
2 complex gave only two bands on SDS-polyacrylamide gel electrophoresis that correspond to the
and
chains (data not shown; see Fig. 3 in Ref. 6 for analogous results) and an absorption spectrum showing the
presence of bound pyridoxal phosphate (see below). Our finding that
each overexpressed mutant
subunit gave a sharp band on
SDS-polyacrylamide gel electrophoresis and was obtained in high yield
indicates that there was no degradation of any of these overexpressed
proteins overall or from the carboxyl end.
and
subunits were purified as described (19). Although the
385-397 mutant protein was expressed as the
subunit by the
plasmid pEBA-6 (
385-397) as described below, this
subunit was
purified as the
2
2 complex for three
reasons as follows: 1) higher yields of
2
2 complex than
subunit are obtained
by the procedures used; 2) a mutant
subunit may be stabilized by interaction with the
subunit; and 3)
2
2 complexes were used in the experiments
reported. The
385-397
subunit was purified as the
2
2 complex after mixing the harvested
cells from two 100-ml cultures (E. coli CB 149/pEBA-6
(
385-397) and E. coli CB 149/pEBA-4A8), which express
the mutant
subunit and the wild type
subunit, respectively.
-T CTC TCT GGC
GGA GAT AAA G-3
; D381S, D381N, or D381Y, 5
-AT
CTC TC
GG
CGC GGA
AAA
GAC AT-3
(n.b. TC
GG
introduces an
AccIII site);
382-397, 5
-C CGC GGA GAT
GAC ATC TTT A-3
; D383A, 5
-GA GAT AAA
ATC TTT ACC
G-3
;
383-397, 5
-C GGA GAT AAA
ATC TTT ACC G-3
;
384-397, 5
-A GAT AAA GAC
TTT ACC GTA C-3
;
385-397, 5
-AAA GAC ATC
ACC GTA CAC G-3
. The
TC
A in the oligomer for
385-397 introduces an
XbaI site. As a result, the mutagenized PCR fragment has two
XbaI sites that prevent recloning into pEBA-10. Therefore, the mutagenized fragment was cloned into pEBA-6 after additional manipulation. The fragment was first inserted into the linear pCRII as
described (6). The product was cut with EcoRI, and the
resulting small fragment was filled in by Klenow and then cut by
SphI to yield a fragment that has an SphI site at
one end and EcoRI/fill in at the other end. This fragment
was then introduced into the SphI and
HindIII/fill in sites of pEBA-6. The mutant plasmids are
designated as parent (mutation) (e.g. pEBA-6 (
385-397) and pEBA-10 (R379Q)). The construction of five mutant proteins with
amino acid replacements at position 382 was as described (6).
2
2 complex in
the conversion of indole (0.2 mM) and L-serine or
-chloro-L-alanine (40 mM) to
L-tryptophan were determined by a spectrophotometric assay
in the presence of an approximately 3-fold excess of
subunit (16,
20). CsCl (0.5 M) was added in some assays where indicated.
The activities of the
2
2 complex in the
reaction (conversion of indole-3-glycerol phosphate to indole and
D-glyceraldehyde 3-phosphate) and in the
reaction (conversion of indole-3-glycerol phosphate and L-serine to
tryptophan and D-glyceraldehyde 3-phosphate) were measured
by a spectrophotometric assay coupled with
D-glyceraldehyde-3-phosphate dehydrogenase in the presence
of excess
subunit (21).
-Scan 1 (Photon Technology
International) dual excitation spectrofluorimeter. Fluorescence
titrations of enzymes with L-serine measured the increase
in fluorescence emission at 510 nm (excitation at 420 nm) upon
incubation of the
2
2 complex (0.26-0.52
µM
pair) with 0.01-10 mM
L-serine at 25 °C (22-24). Kd values
for L-serine were obtained by hyperbolic curve fitting of
the change in fluorescence
F using Equation 1:
Kd values for
(Eq. 1)
-chloro-L-alanine were obtained from the competitive
inhibition by
-chloro-L-alanine of the fluorescence of
the enzyme complexes with L-serine. In Method 1, several
concentrations of
-chloro-L-alanine were used (0, 0.2, 0.5, 1, and 3 mM for the wild type and 0, 10, 20, 40, and
60 mM for the K382N
2
2 complex), and Ki values were obtained by linear fit
of Kobs/Kd versus
[
-chloro-L-alanine] using Equation 2:
In Method 2, a single concentration of
(Eq. 2)
-chloro-L-alanine was used (0.5 or 4 mM),
and Ki values were obtained from double-reciprocal
plots of
F versus [L-serine] using Equation 3:
(Eq. 3)
Enzyme solutions (2 mg/ml
2
2 complex in ~10 mM sodium
N,N-bis(2-hydroxyethyl)glycine containing 0.1 mM pyridoxal phosphate, 0.1 M potassium
phosphate, and 1 mM EDTA at pH 7.8) were incubated in
Eppendorf tubes in a water bath at 60 °C for various times. Tubes
were chilled to 4 °C for 30 min or more and then centrifuged. Aliquots of supernatant solutions were assayed for enzymatic activity in the
reaction with indole and L-serine in the
presence of a 3-fold excess of wild type
subunit.
To investigate the functional and structural roles of the
C-terminal region of the tryptophan synthase subunit, we have engineered 12 point mutations of the conserved residues Arg-379, Asp-381, Lys-382, and Asp-383 and 4 C-terminal truncations. We did not
investigate the effects of mutations altering the conserved residues
Gly-378 and Gly-380 in this work because these residues do not interact
with other residues and have not been implicated previously as
important residues. Initial studies on five mutant proteins altered at
position 382 have been reported (6). The engineered mutant
subunits
were all expressed in high yield and purified as stable
2
2 complexes (see "Experimental
Procedures").
Interaction of the wild type and
subunits
stimulates the catalytic activity of the
subunit in the
reaction (Equation 4) about 50-fold (see Ref. 1 and footnote to Table
I).
![]() |
(Eq. 4) |
|
Table I gives the specific activities of the wild type
and mutant 2
2 complexes in the synthesis
of L-tryptophan from L-serine and indole (
reaction, Equation 5) and from L-serine and
indole-3-glycerol phosphate (
reaction, Equation 6).
![]() |
(Eq. 5) |
![]() |
(Eq. 6) |
We have arbitrarily classified the mutant
2
2 complexes on the basis of the effects
of the mutations on the activity in the
reaction, the reaction of
L-serine with indole. "Class I" enzymes, which have
mutations at positions 379 and 381 or truncations after residue 383 (384-397 or 385-397), exhibit significant activity (1-38% of wild
type). "Class II" enzymes, which have mutations at positions 382 or
383 or truncations including residues 382 or 383 (382-397 or
383-397), exhibit very low activity (<1% of wild type). The absence
of L-serine stimulation of indole-3-glycerol phosphate
cleavage by Class II enzymes (Ratio
reaction/
reaction = ~1 in Table I) can be attributed to the lack reaction of
L-serine at the
site.
Some mutant forms of tryptophan synthase have considerably higher
activity in the overall reaction than in the individual
reaction (25, 26). These results were partly due to a reduced association of the mutant
subunit with the
subunit; addition of
indole-3-glycerol phosphate during catalysis of the
reaction or
reaction increased association and activity. The finding that
the activity of each mutant protein in Class I is quite similar in the
and
reactions (Table I) shows that Class I mutations have no
effects on association between the
and
subunits that can be
detected by these assays of enzymatic activity.
Table II compares
the activities of the wild type and mutant enzymes in the reaction of
-chloro-L-alanine with indole to activities in the
reaction of L-serine with indole. It is striking that all
of the mutant enzymes, except the Class I enzymes with mutations at
position 379, have rather high activities with
-chloro-L-alanine (42-140% of the wild type enzyme).
Thus, the Class II mutations, which lead to loss of activity with
L-serine and indole, do not prevent the analogous reaction
with
-chloro-L-alanine. Consequently, the substrate
specificity ratio, which is defined as the ratio of activity with
-chloro-L-alanine and indole to activity with L-serine and indole, is much higher for the Class II
enzymes (25-500) than for the wild type
2
2 complex (0.24) or the Class I mutant proteins (0.11 to 8.7).
|
Table III
compares the binding constants of L-serine and
-chloro-L-alanine for the wild type and several mutant
2
2 complexes. The results were obtained
by determining the effect of L-serine concentration on the
change in fluorescence emission at 510 nm in the presence or absence of
fixed concentrations of
-chloro-L-alanine, a competitive
inhibitor that does not form a fluorescent external aldimine (22-24).
The Kd values of L-serine and
-chloro-L-alanine are similar for all of the enzymes
except K382N. Although the binding constant of L-serine (3 mM) for the K382N
2
2 complex is markedly elevated, this enzyme should be saturated by the
concentration of L-serine (40 mM) used in the
assay. The K382N
2
2 complex exhibits high
activity with
-chloro-L-alanine and indole even though
the Kd for
-chloro-L-alanine (78 mM) is higher than the concentration of
-chloro-L-alanine (40 mM) used in the assay.
Thus, the effects of the Class II mutations on substrate specificity
are not attributable to changes in the binding of L-serine
or of
-chloro-L-alanine.
|
Because CsCl activates
certain mutant 2
2 complexes (11),
including those having single amino acid replacements of
subunit Lys-382 (6), we have also determined activity in the
reaction in
the presence of 0.5 M CsCl (Table II). CsCl activates all
of the mutant
2
2 complexes that have low
activity but has little effect on the R379Q and R379K
2
2 complexes that have relatively high
activities. CsCl results in a striking 16-fold activation of the R379P
2
2 complex, which has a much lower
activity than R379Q and R379K under standard conditions. CsCl also
activates the wild type
subunit (11).
The wild type
2
2 complex and
subunit catalyze a
-replacement reaction of L-serine with
-mercaptoethanol that proceeds through a series of pyridoxal
phosphate-substrate intermediates that have characteristic absorption
spectra (see Fig. 3 for structures and abbreviations)
(27, 28). Whereas an equilibrium mixture of these intermediates
accumulates with the wild type
2
2
complex, the E-Ser intermediate predominates with the wild
type
subunit and most of the mutant
2
2 complexes (data not shown). Conversion of E-Ser to E-AA is rate-limiting with the wild
type
subunit (29). Because Cs+ and
NH4+ alter the rate of this conversion
by the
subunit (29) and alter the rates and spectroscopic
properties of some mutant enzymes (11), we have determined the
absorption spectra obtained upon reaction of L-serine and
of L-serine and
-mercaptoethanol in the presence of 0.5 M CsCl (Fig. 3 and Table II). Fig. 3, A and B, shows the spectra obtained in the presence of 0.5 M CsCl with the R379Q
2
2
complex (a representative of Class I) and with the
383-397
2
2 complex (a representative of Class
II), respectively. The three spectra obtained with the R379Q
2
2 complex are essentially identical to
those of the wild type
2
2 complex under
the same conditions. The enzyme alone exhibits a major peak centered at 410 nm due to the internal aldimine (E). Reaction with
L-serine yields a complex spectrum with a major peak
centered at 340-350 nm, which is ascribed to the external aldimine
between pyridoxal phosphate and aminoacrylate (E-AA).
Reaction of L-serine and
-mercaptoethanol yields a major
peak at 468 nm, which is ascribed to a quinonoid (E-Q)
intermediate formed upon addition of
-mercaptoethanol to the
aminoacrylate intermediate. The
383-397 enzyme alone also exhibits
a peak at 410 nm (E). Addition of L-serine
yields a major peak at 424 nm which is ascribed to the external
aldimine between pyridoxal phosphate and L-serine
(E-Ser). The spectrum is unchanged by the further addition
of
-mercaptoethanol.
Table II presents the wavelength of maximum absorbance in the presence
of L-serine or of L-serine plus
-mercaptoethanol for the wild type
2
2
complex and
subunit and for each mutant
2
2 complex in the presence of CsCl. In
general, the three spectra for each mutant
2
2 complex in Class I were similar to
those of the wild type
2
2 complex,
whereas the spectra for each mutant
2
2
complex in Class II were similar to those of the
383-397
2
2 complex and of the wild type
subunit. Thus, CsCl partially restores the wild type spectral
properties of the Class I mutant
2
2
complexes but not of the Class II mutant
2
2 complexes.
Addition of a synthetic peptide corresponding to
residues 1-9 of the tryptophan synthase subunit partially restores
the activity of a mutant
subunit having residues 1-9 deleted (30). To test whether a peptide corresponding to the deleted helix 13 could
restore the activity and spectral properties of the mutant
subunit
having residues 383-397 deleted, we mixed the
383-397
2
2 complex (7.8 µM) with
the peptide corresponding to residues 383-393 (1.05 mM) in
Buffer B in the absence and presence of 0.5 M CsCl at
25 °C for 90 min. Assays of the activity with L-serine and indole in the presence of CsCl (as described in Table II in the
absence of peptide) showed that addition of the peptide had no effect
on the activity. Similarly, the peptide did not affect the absorption
spectra of this mutant enzyme and its enzyme-substrate complexes either
in the absence of CsCl or in the presence of CsCl (as described in Fig.
3B in the absence of peptide).
Fig. 4 shows the rates
of thermal inactivation of the wild type and three mutant subunits
upon incubation of the corresponding
2
2
complexes at 60 °C. Because the
subunits in the
holo-
2
2 complexes undergo thermal
inactivation at 60 °C (31), the activities of the
subunits were
determined in the presence of added excess
subunit. The results
show that the
subunits in the two C-terminal truncation mutant
2
2 complexes examined (
383-397 and
384-397) were much more thermolabile than the wild type
subunit. The D381Y
subunit was somewhat more thermolabile than the
wild type
subunit.
The experiments described above were aimed at assessing the
functional roles of the conserved and variable residues and of helix 13 in the C-terminal region of the subunit. Because the C-terminal
residues 378-397 are outside of the core region of the C-terminal
domain (5) and are not conserved in related enzymes (7), these residues
may have evolved to have special functions, such as stabilizing the
2 dimer.
The four subunit C-terminal truncations and 12 point mutations were
all expressed in high yield as stable
2
2
complexes ("Experimental Procedures" and Ref. 6), contained bound
pyridoxal phosphate that forms enzyme-substrate intermediates (Fig. 3
and Table II), and activated indole-3-glycerol phosphate cleavage by
the
subunit (Table I). Thus, the residues deleted (
382-397) or
altered (379, 381, 382 and 383) are not required for overall folding of
the
subunit, for association of the
subunit with the
subunit to form an
2
2 complex, or for
activation of the
subunit. These results document the structural
integrity of these mutant
subunits. The
subunit C-terminal
helix 13 may serve a structural role because the mutant proteins having
residues 383-397 or 384-397 deleted were much more thermolabile (Fig.
4).
Residues 379 and 381-397 are not essential for catalysis because all
of the mutant enzymes exhibit some activity in the reaction of
L-serine with indole (0.01-38% of wild type) and quite
significant activity in the reaction of
-chloro-L-alanine with indole (10-140% of wild type)
(Table II). We have divided the mutant proteins into two classes, I and
II, based on activity in the reaction of L-serine with
indole. Class I mutant proteins are more similar to the wild type
enzyme than Class II mutant proteins in activity with
L-serine and indole, in substrate specificity, and in
spectroscopic properties (Tables I and II and Fig. 3). The results with
Class I mutant proteins show that Arg-379, Asp-381, and residues
384-397 are not very important for catalytic activity. Although
substitution of Arg-379 by Lys or Gln has minor effects on the
catalytic properties, substitution by Pro (R379P) has more drastic
effects. This result is consistent with the finding that E. coli cells having this mutation in the trpB gene are
unable to convert indole to tryptophan (32). Our results do not explain
why the R379P mutant cells were found to produce more indole than cells
with a trpB mutation at position 382 (32). The activity in
the
reaction is in fact lower than that of any of the other mutant
proteins. The proline replacement may alter the backbone geometry and
have a more deleterious effect on the conformation of the
subunit
than the more conservative Lys and Gln replacements.
Class II mutant proteins have extremely low activities in the reaction
with L-serine and indole but have activities close to that
of wild type in the reaction of -chloro-L-alanine with indole (Tables I and II). These mutant proteins all have a substitution or deletion of Lys-382 or of Asp-383. These results support and extend
observations that the mutation of Lys-382 results in inactivation (1,
32, 33). The D383A and
383-397 mutant proteins lack the conserved
residue Asp-383. The finding that these mutant proteins have extremely
low activity in the reaction of L-serine and indole provides the first evidence that Asp-383 is very important for the
catalysis of this reaction. The effects of the Class II mutations on
activity in the reaction of L-serine with indole are not
attributable to changes in the binding of L-serine because
the Kd values of L-serine for the mutant
enzymes (Table III) are all lower than the concentration of
L-serine (40 mM) used in the assay. The
importance of Lys-382 for tight binding of L-serine and
-chloro-L-alanine may be attributable to formation of a
salt bridge between Lys-382 and Glu-350 (see Fig. 1B and
Ref. 6). This salt bridge could stabilize the substrate binding site.
An additional role for Lys-382 in a substrate-induced conformational
change is discussed below.
Class II mutant proteins exhibit altered
substrate specificity and an altered distribution of enzyme-substrate
intermediates in the reaction of L-serine and
-mercaptoethanol (Table II and Fig. 3). These results are now
discussed in relation to previous studies of the conformational states
of tryptophan synthase. Dunn and co-workers (12-15) have drawn upon
data from a number of investigations of tryptophan synthase by
different groups of investigators to formulate a model depicting
ligand-mediated conformational changes that occur during the course of
the
reaction. The model postulates that the
and
subunits
undergo transitions between open and closed conformations which
function to coordinate the catalytic activities and promote diffusion
of the indole intermediate. Recent investigations using
8-anilino-1-naphthalenesulfonate binding as a probe have identified
three distinct conformations of the
2
2
complex associated with different liganded states of the
and
subunits (13). The conformation of the
subunit stabilized by
E-AA and E-Q has been designated as closed,
whereas the conformation stabilized by E-Ser and
E-S-hydroxyethylcysteine has been designated as
open (see diagram at the top of Fig. 3) (13).
In the reaction of L-serine with -mercaptoethanol, the
E-AA (closed) intermediate predominates in the
wild type and Class I mutant enzymes, whereas the E-Ser
(open) intermediate predominates in the Class II mutant
enzymes (Table II and Fig. 3). This result indicates that the mutations
hinder the ligand-mediated transition from an open to a closed
structure either by stabilizing the open structure or by destabilizing
the closed structure. The E-Ser intermediate also
predominates in several other
2
2
complexes having mutations in the
subunit (6, 11, 16). We have postulated that the wild type
subunit and these mutant
2
2 complexes have a conformation (termed
"open") that results in a poor alignment of the weak
hydroxyl leaving group of L-serine for protonation and
-elimination. These enzymes may have much higher activity with
-chloro-L-alanine because this substrate has a strong
leaving group that does not require protonation. The finding that
addition of CsCl (0.5 M) activates a number of open mutant
proteins and the wild type
subunit suggests that Cs+
may stabilize a more active alternative conformation of these enzymes
(6, 11). CsCl also increases the very low activities of the D383A,
382-397, and
383-397 mutant proteins 10-80-fold (Table II).
CsCl may stabilize the more active, closed conformation of the
subunit and partially shift the equilibrium from the open to the closed
form (11).
Fig. 1, A and B, shows the
locations of residues that have been altered or deleted in the present
investigation in the three-dimensional structure of the wild type
2
2 complex. The region containing altered
residues (residues 378-393) is shown in red. The C-terminal residues 394-397 are not clearly visible in the structure and are not
shown. The C-terminal helix 13 from each
subunit is located near
the
/
interaction site and protrudes from the center of the
subunit into solvent. Three residues in helix 13 are involved in
/
interactions (Fig. 1B). These interactions are Ile-384 to Pro-144, His-388 to Phe-147, and Leu-391 to Phe-147. Two of
the conserved residues mutated (Arg-379 and Asp-381) also make
/
interactions: the two Arg-379 residues are stacked with each other;
Asp-381 interacts with Arg-148. Lys-382 forms an internal salt bridge
with Glu-350 of the same
subunit. Although the four C-terminal
truncations remove the three
/
interactions made by Ile-384,
His-388, and Leu-391, the mutant
subunits all form stable
2
2 complexes. Thus, disruption of
interactions between six pairs of residues in the
2
2 complex is not sufficient to cause
disruption of the
/
interface. Similarly, single amino acid
substitution of Arg-379 or Asp-381 does not cause disruption of the
/
interface.
How are ligand-mediated conformational changes in tryptophan synthase
related to structural changes? Crystallographic investigations are only
beginning to reveal structural effects of ligands. Recent studies
reveal that exchange of K+ or Cs+ for
Na+ induces local and long range changes in the
three-dimensional structure of the tryptophan synthase
2
2 complex (10). Studies of a mutant form
of the enzyme (
K87T) having an external aldimine of
L-serine at the
site and indole-3-propanol phosphate or
DL-
-glycerol 3-phosphate at the
site show
conformational changes that may be related to formation of the closed
conformation in solution.2 Conformational changes observed
in these structures of the residues investigated here include movements
of Arg-141 toward Thr-386 and of Gln-142 toward Lys-382 and Asp-383.
The possible involvement of these interactions in the closed
conformation may explain why the mutation or deletion of Asp-383 or of
Lys-382 in Class II mutant enzymes appears to destabilize the closed
form of the enzyme and prevent the open to closed transition.
Because proteins are stabilized by a large network of interactions,
mutations are quite likely to interfere with this complex network and
destabilize the protein or alter the conformation of the enzyme needed
for optimal activity (34). Some proteins are stabilized by better
attachment of the N and C termini to the rest of the molecule to
prevent "fraying" (34). Deletion of one of these termini may remove
these stabilizing interactions and promote fraying. Although the
C-terminal helix 13 of the subunit is partially exposed to solvent,
it does make several interactions in the
/
interface. The removal
of helix 13 may destabilize the
subunit to heat and loosen
/
interaction by removing these interactions as discussed above. Addition
of a synthetic peptide corresponding to residues 383-393 did not
restore the catalytic activity or the spectroscopic properties of the mutant
subunit having residues 383-397 deleted.
Our results show that helix 13 is important for
thermal stability and that residues 379, 381, 382, and 383 in the C
terminus of the subunit are important, but not essential, for
catalytic activity. Lys-382 and Asp-383 appear especially important for stabilization of the closed conformation of the enzyme that has optimal
activity with L-serine.
We express our appreciation to Drs. Charles Yanofsky and Peter McPhie for many thoughtful comments and helpful suggestions. We thank Dr. David R. Davies for crystallographic facilities and encouragement. We thank Dr. Roger S. Rowlett for help with data analysis.