(Received for publication, December 19, 1994)
From the
This work is aimed at understanding subunit assembly in the
tryptophan synthase complex and the
importance of the internal aldimine between pyridoxal phosphate and
lysine 87 of the
subunit of tryptophan synthase for
subunit association. We utilize a mutant form of the
subunit that is unable to form the internal aldimine because
lysine 87 is replaced by threonine (K87T). The K87T
complex is inactive in reactions
catalyzed by the
subunit but retains activity in the
reaction catalyzed by the
subunit. We find that dialysis removes
pyridoxal phosphate much more rapidly from the K87T
subunit and
complex than from
the wild type counterparts. Activity measurements, gel filtration, and
subunit interchange experiments show that the
subunit dissociates
more readily from the K87T
subunit than from the wild
type
subunit. The reaction of L-serine to
form an external aldimine with pyridoxal phosphate at the active site
of the K87T
subunit markedly increases the affinity
for the
subunit and slows removal of pyridoxal phosphate by
dialysis. We propose that the external aldimine between L-serine and pyridoxal phosphate bridges the N-domain and the
C-domain in the K87T
subunit. This interdomain bridge
may mimic the internal aldimine bond in the wild type
subunit and stabilize pyridoxal phosphate binding. The
interdomain bridges formed by the internal aldimine with the wild type
subunit and by the external aldimine with L-serine in the K87T
subunit may further
stabilize interaction with the
subunit because the
/
interaction site contains residues from both N- and C-domains of the
subunit.
The bacterial tryptophan synthase complex (EC 4.2.1.20) is a useful system for investigating
protein-protein interaction and the mechanism of subunit assembly (for
reviews see (1, 2, 3, 4, 5, 6) ).
The
complex dissociates into two
subunits and one
subunit. The monomeric
subunit catalyzes the
reaction. The dimeric
subunit contains one pyridoxal phosphate at each active site and
catalyzes the pyridoxal phosphate-dependent
reaction. The
complex catalyzes the
and
reactions and the overall
reaction, which is formally
the sum of the
and
reactions.
The three-dimensional structure of the tryptophan synthase
complex from Salmonella
typhimurium(7) reveals that the
and
subunits are arranged in a nearly linear
order. The active sites of the
and
subunits are
25
Å apart and are connected by a tunnel that passes through the
/
interaction site and between the two structural domains of
the
subunit. The pyridoxal phosphate coenzyme is
located at the interface between the two structural domains (N-domain
and C-domain) of the
subunit at one end of the tunnel
and interacts with residues from both domains. The carbonyl group of
pyridoxal phosphate forms an internal aldimine with Lys-87 in the
N-domain. The phosphate group of the coenzyme forms hydrogen bonds with
Gly-232, Gly-233, Gly-234, Ser-235, Asn-236, and Ala-237 in the
C-domain. The pyridine ring nitrogen atom, N1, is close to the
sulfhydryl of Cys-230 and within hydrogen bonding distance of the
hydroxyl of Ser-377, both residues in the C-domain.
Early studies of
the association of the and
subunits of
tryptophan synthase using sucrose gradient centrifugation showed that
pyridoxal phosphate partially stabilized the
complex(8) . The
apo-
complex (minus pyridoxal
phosphate) was totally dissociated, the
holo-
complex (plus pyridoxal
phosphate) was partially dissociated, and the holo-
complex in the presence of L-serine was stable. Pyridoxal phosphate and L-serine
also increased the apparent subunit association constants measured
under assay conditions(8) . The reaction of L-serine
with the
complex resulted in a
400-fold increase in affinity of the
subunit for the
subunit(9) .
In the present work we ask whether
binding pyridoxal phosphate per se increases subunit affinity
or whether formation of the internal aldimine with subunit lysine 87 is required. To answer this question, we use a
mutant form of the
subunit having the lysine 87
replaced by threonine (K87T)(10) . Studies with the K87T
complex demonstrated that lysine 87
serves critical roles in transimination, catalysis, and product
release(11) . The K87T
complex is inactive in the
reaction but retains activity in
the
reaction. The mutant enzyme binds pyridoxal phosphate as the
free aldehyde and forms Schiff base intermediates (external aldimines)
with L-serine (K87T-Ser) or L-tryptophan (K87T-Trp)
at the
site. Crystallographic analyses of the external aldimines
formed by the K87T
complex with L-serine and L-tryptophan have been carried out at
2Å resolution (
)and have localized the substrate
and product binding sites in the
subunit(12) . The crystallographic results show that L-serine and L-tryptophan are bound between the N-
and C-domains and interact with some residues of the N-domain. The
results presented here provide evidence that formation of interdomain
bridges by pyridoxal phosphate-aldimine intermediates stabilizes
subunit association and stabilizes pyridoxal phosphate binding.
Figure 1:
Effect of time of dialysis on the
pyridoxal phosphate content of various forms of tryptophan synthase.
Solutions of the holo- complex (A) and of the holo-
subunit (B) at
1 mg/ml were dialyzed against Buffer B at 4 °C. Aliquots were
analyzed at intervals for protein concentration and for pyridoxal
phosphate content. Curve1, wild type; curve2, K87T; curve3, K87T-Ser
complex.
Figure 2:
Superose gel filtration of various forms
of wild type and mutant (K87T) tryptophan synthase. A, wild
type holo- complex (1 mg), holo
subunit (1 mg), and
subunit (0.38 mg) were
injected in separate runs. B, wild type
subunit (0.42
mg) was preincubated for 3 h at 23 °C with approximately equimolar
reduced
subunit (0.66 mg), K87T
subunit (0.6 mg), or K87T
subunit plus 50
mML-serine before injection in separate runs. C, wild type
subunit was preincubated with 2 eq of
protomer to give the
complex; the K87T
complex was injected as in A.
Mixing the subunit
with 2 or more eq of holo-
protomers results in a stable
complex that can be observed by sucrose
centrifugation (8, 22) or
ultracentrifugation(21) . The wild type holo-
complex elutes at 10.0 ml in our high performance gel filtration
system (Fig. 2C). Gel filtration of the K87T
holo-
complex yields two peaks at
positions characteristic of the
complex and of
the
subunit (Fig. 2C). (
)The identity
of these two species was confirmed by assay of activity in the
reaction and by SDS-gel electrophoresis (data not shown). Thus the K87T
holo-
complex partially dissociates
under these conditions. Similar results were obtained with a
reconstituted K87T holo-
complex
prepared by mixing the K87T holo-
subunit and 2 eq of
wild type
subunit (Fig. 2B). As controls,
reconstituted
complexes were also
prepared by mixing holo-
subunit or reduced
subunit with 2 eq of
subunit. The reconstituted
reduced
complex (Fig. 2B) and holo-
complex (data not shown) each yielded a major peak at the
position of
complex. Gel filtration
of the reconstituted K87T holo-
complex in the presence of L-serine also yielded a major
peak at the position of the
complex.
Thus L-serine largely prevents dissociation of the K87T
holo-
complex to
and
subunit under these conditions.
Fig. 3shows the enzymatic activity of the wild type
subunit in the
reaction obtained upon titration with
increasing amounts of wild type holo-
subunit, reduced
subunit, and K87T holo-
subunit.
Titrations with the reduced and K87T holo-
subunit
were also carried out in the presence of L-serine. It is
noteworthy that the maximum specific activity of the
subunit in
the
reaction (see S in Table 1) varies significantly when
the
subunit is complexed with different species of
subunit. Early studies also observed that
complexes containing reduced
subunit from Escherichia coli had 2-fold
greater activity than the wild type
complex in the
reaction(23) . It is known that the
activity of the
subunit is sensitive to the conformational state
of the
subunit because ligands that bind to the
active site of the
subunit alter the kinetics of
reaction at the active site of the
subunit
25 Å
distant(13, 19, 24, 25, 26, 27, 28) .
Reduction of pyridoxal phosphate at the active site of the
subunit (reduced
) and substitution of threonine
for lysine 87 (K87T
) probably cause alterations in
the conformation of the
subunit that are communicated
to the active site of the
subunit and alter the activity of the
subunit.
Figure 3:
Titration of the subunit with
various
subunits. The activity of the
subunit
(0.88 µM) in the
reaction was determined in the
presence of various amounts (0-2.64 µM) of the wild
type
subunit (
), the reduced
subunit (
), or the K87T
subunit
(
). Data in the presence of 40 mML-serine was
determined with various amounts of the reduced
subunit (
) or the K87T
subunit (
).
Specific activity expressed in units/mg
subunit is plotted versus the molar ratio of
/
. Data were analyzed as
described in the text and fit to models 1-3 (see Table 2).
Derived apparent dissociation constants are shown in Table 2.
The data obtained in Fig. 3and additional
data (not shown) for titration with the apo- subunit
were analyzed using the PC-MLAB program (Civilized Software, Bethesda,
MD) and fitted to three possible models (Table 1): 1) the
subunit binds two
subunits with equal
affinities (K
= K
; both bound
subunits are
active); 2) the
complex is active,
whereas the
complex is inactive; 3) the
subunit binds two
subunits with different
affinities (both bound
subunits are active). Model 3 gave the
best fit (as judged by the sum of squares) for the wild type and
reduced
subunits. (
)The affinities of the
first
subunit for the holo-
subunit are too high
to measure by this method (K
= <0.01
µM; Table 1). The dashedline in Fig. 3shows the theoretical curve for stoichiometric binding of
two
subunits to the reduced
dimer where the K
= <0.01 µM for both
subunits. Analysis showed that the second
subunit binds to the
reduced
subunit and to the wild type holo-
subunit with K
= 0.07-0.08
µM (Table 1). In marked contrast, the K87T
holo-
subunit exhibits a gradual titration curve that
indicates much weaker and equal affinities for both
subunits with K
=
0.4 µM (Table 1). In the presence of L-serine, the K87T
holo-
subunit again exhibits a sharp biphasic
titration curve, with the second K
=
0.05 µM (Table 1). This result shows that L-serine greatly strengthens the association of the K87T
holo-
subunit with the
subunits. Addition of L-serine to the reduced
subunit has no
effect. This is to be expected because the reduced
subunit does not react with L-serine. Addition of L-serine to the wild type holo-
subunit in
the presence of the
subunit and indole-3-glycerol phosphate
results in the overall
reaction which is about 20 times
faster than the
reaction (data not shown). Association of the
and
subunits under the conditions of the
reaction is reported to be very tight (K
= 0.4
10
µM)(8) . The apo-
subunit
exhibits much weaker and equal affinities for both
subunits with K
=
0.3 µM (Table 1).
Figure 4:
Kinetics of dissociation of the wild type
complex. Dissociation was initiated
by addition of excess reduced
subunit in the absence (curve1) or presence (curve2) of
40 mML-serine or by addition of the K87T-Ser
subunit complex in the absence (curve3) or presence (curve4) of 40 mM of L-serine. Activity measured at intervals in the
reaction is shown relative to the activity of the
complex alone (see
``Experimental Procedures'').
Our plan to study
the rate of dissociation of the wild type complex upon addition of excess K87T
subunit
was precluded by our inability to obtain completely serine-free K87T
subunit. Trial experiments (not shown) indicated that
the small fraction (
10%) of the K87T
subunit
that bound L-serine (K87T
-Ser) was much more
effective than the serine-free fraction (K87T
) in
binding
subunit and causing dissociation of the wild type
complex. Consequently we determined
the rate of dissociation of the wild type
complex upon addition of the isolated K87T
-Ser
complex in the absence of excess L-serine (curve3) and in the presence of excess L-serine (curve4). Addition of K87T
-Ser (curve3) results in more rapid dissociation of the
wild type
complex than does addition
of the reduced
subunit (curve1).
Thus, K87T
-Ser binds the
subunit more rapidly
than does reduced
. Addition of excess L-serine (curve4) tightens the association
of the wild type
complex and
decreases the rate of dissociation of the
complex.
The rate of dissociation of an
complex containing inactive
subunit can be measured by determining the increase
in activity upon addition of excess wild type
subunit. This method has been used previously for the reaction of
reduced
complex with excess wild
type
subunit (9) as illustrated in Fig. 5, curve1. We have determined activity
in the
reaction because the free
subunit
has no activity in the
reaction, whereas the
subunit does have some activity in the
reaction that would
give a high blank value. Addition of excess wild type
subunit to the K87T
complex
results in 40% activity within the mixing time (Fig. 5, curve2) followed by a slower increase in activity.
When the K87T
-Ser complex was mixed
with wild type
subunit in the presence of 40 mML-serine (Fig. 5, curve3), the
rate of activation was much slower in the first 5 min than in curve2, but full activity was achieved after about 30 min. The
reaction of the K87T
-Ser complex
with excess
subunit (Fig. 5, curve4) showed a decreased rate of activation similar to that
in curve3. The results indicate that the first
subunit in the K87T
complex
dissociates readily in the absence of L-serine but more slowly
in the presence of L-serine. The very slow rate of subunit
interchange with the K87T
-Ser
complex (Fig. 5, curve4) implies that the
K87T
-Ser dissociates very slowly.
The slow rate of subunit interchange with the K87T
complex (Fig. 5, curve2) may result from the
3% contamination of the
enzyme with L-serine (see ``Experimental
Procedures'').
Figure 5:
Kinetics of dissociation of the reduced
complex and K87T
complex. Excess wild type
subunit was added to initiate dissociation of the
reduced
complex (curve1), of the K87T
complex in the absence (curve2) or presence (curve3) of 40 mML-serine, or of
the K87T-Ser
complex (curve4). Activity measured at intervals in the
reaction is shown relative to the activity of the wild type
complex (see ``Experimental
Procedures'').
Our studies of a mutant form of tryptophan synthase, which is
unable to form the internal aldimine between subunit
Lys-87 and pyridoxal phosphate demonstrate that the internal aldimine
is very important for stabilizing cofactor binding and subunit
association.
Binding of
pyridoxal phosphate and pyridoxal phosphate analogues to the wild type
subunit and
complex has been studied
extensively(29, 30, 31, 32) .
Pyridoxal phosphate is bound cooperatively to the apo-
subunit and noncooperatively to the
apo-
complex. The slow rate of
removal of pyridoxal phosphate from the wild type
complex by dialysis probably results
from the very slow reversal of the final isomerization step in
pyridoxal phosphate binding(32) . The rate of removal of
pyridoxal phosphate from the
subunit and
complex by dialysis has not been
compared previously. However, we have reported that the pyridoxal
phosphate oxime is much more readily removed from the
subunit than from the
complex(15) . We have suggested that interaction of the
subunit with the
subunit may stabilize the
interaction between the N- and C-domains of the
subunit and thus slow removal of pyridoxal phosphate and
pyridoxal phosphate derivatives that are bound between the two
domains(2) .
Subunit
interchange experiments show that the rate of dissociation of the
holo- complex by reduced
subunit is greatly decreased in the presence of L-serine (Fig. 4, curve 2 versus1). Thus, reaction of L-serine with the wild type
complex decreases the rate of dissociation (9) and
tightens association (strong association in Table 2)(8) .
Dissociation of the holo-
complex by
K87T
-Ser is also greatly reduced in the presence of L-serine (Fig. 4, curve 4 versus3).
The similarity of the results with reduced
and with
K87T
-L-Ser in Fig. 4supports the
classification of these two enzymes in the same group (intermediate
association) in Table 2. The finding that the K87T
complex dissociates readily in the
absence of L-serine but more slowly in the presence of L-serine (Fig. 5) implies that the external aldimine
with L-serine increases association from weak to intermediate (Table 2).
The dissociation constants for various forms of the
complex ( Fig. 3and Table 1) are apparent dissociation constants since they are
measured in the presence of a substrate (indole-3glycerol phosphate)
that may alter subunit affinity. Our new methods of analysis of the
titration curves shows that most forms of the
subunit
have higher affinity for the first
subunit than for the second
subunit. This result is consistent with other evidence that the
wild type
subunit binds the
subunit with
negative cooperativity(9, 33) . The results in Fig. 3and Table 1demonstrate that L-serine
increases the affinity of the K87T
subunit for the
subunit by 1-2 orders of magnitude.
Our previous finding that the
K87T complex has very weak
ellipticity, whereas the external aldimine with L-serine has
much greater ellipticity(20) , indicates that pyridoxal
phosphate becomes more rigidly oriented when its carbonyl group forms
an external aldimine with L-serine. The increased rigidity
results from the binding of L-serine to the active site. The
reduced
complex is stabilized by the
covalent link between pyridoxal phosphate and Lys-87. The interdomain
bridges formed by the internal aldimine with the wild type
subunit, by reduced phosphopyridoxyl-lysine in the reduced
subunit, and by the external aldimine with L-serine in the K87T
subunit may stabilize
interaction with the
subunit because the the
/
interaction site contains residues from both the N-domain and the
C-domain.
The substrate of an enzyme often binds in a cleft between
two domains and can be considered to bridge the two domains. Example
enzymes include aspartate aminotransferase(34) , citrate
synthase(35) , hexokinase(36) , and human
-thrombin(37) . Salt bridges and disulfide bonds may also
form bridges between two enzyme domains and stabilize the enzymes to
unfolding by heat and denaturants.
In conclusion, our work has shed
light on aspects of interdomain interaction that stabilize both the
subunit and its interaction with the
subunit.
The internal and external aldimine intermediates formed by pyridoxal
phosphate stabilize the interaction of pyridoxal phosphate with
tryptophan synthase and stabilize the enzyme structure. Recent studies
on the thermal inactivation (38) and thermal unfolding (39, 40, 41) (
)also demonstrate
that pyridoxal phosphate stabilizes the
subunit and
complex of tryptophan synthase to
unfolding and increases the linkage (cooperativity) between unfolding
domains in the
subunit.