Tryptophan synthase catalyzes the last two reactions in the
biosynthesis of tryptophan (for reviews, see Miles(1979, 1991, 1995)).
The
subunit catalyzes the cleavage of indole 3-glycerol phosphate
(IGP) (
)to indole and glyceraldehyde 3-phosphate (G3P)
(
reaction), while the
subunit catalyzes the condensation of
indole with serine in a reaction mediated by pyridoxal phosphate (
reaction). The physiologically important reaction is termed the

reaction and involves the conversion of IGP and serine to
tryptophan and water (Demoss, 1962; Creighton, 1970; Matchett, 1974).
These reactions are shown in Fig. SI. Tryptophan synthase, an



tetrameric complex, is considered a
classic example of an enzyme that is thought to exhibit substrate
channeling: a process in which a metabolic intermediate is directly
transferred from one active site to another without free diffusion. The
solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provides physical evidence for a
25-Å hydrophobic tunnel which connects the
and
active
sites (Hyde et al., 1988). In this case, the metabolic
intermediate (indole) would be transferred from the active site of the
subunit to the active site of the
subunit through the
connecting tunnel. Using rapid reaction kinetics, we have previously
established that indole is indeed channeled and have identified three
essential kinetic features which govern efficient channeling. Previous
rapid kinetic analysis of tryptophan synthase has shown that the
product of the
reaction, indole, is transferred to the
site
where it reacts with serine to form tryptophan (Anderson et
al., 1991; Lane and Kirschner, 1991; Brzovic et al.,
1992). The transfer of the indole intermediate is highly efficient such
that indole cannot be observed in a single enzyme turnover of the

reaction. The efficient channeling of indole is a
consequence of three features of the reaction kinetics. 1) The rate of
diffusion of indole through the channel is very fast; 2) the reaction
of indole to form tryptophan at the
site is fast and largely
irreversible; and 3) the reaction of serine at the
site modulates
the formation of indole at the
site or in the tunnel such that
indole is not produced until serine has reacted with pyridoxal
phosphate to form the highly reactive aminoacrylate (Anderson et
al., 1991). This intersubunit communication keeps the
and
reactions in phase so that indole does not accumulate at the
site. The combination of these three aspects of the reaction
kinetics leads to efficient channeling of indole by maintaining a low
concentration of indole bound to the enzyme. Accordingly, this model
for channeling of the indole intermediate predicts that blocking or
impeding the passage between active sites might allow detection of
indole during a single enzyme turnover. One of the residues lining the
tunnel is
C170. Site-directed mutagenesis of the
C170 to Phe
and Trp has been used to construct mutants of tryptophan synthase in
which the tunnel is restricted (Schlichting et al., 1994;
Ruvinov et al., 1995). In this report we describe the kinetic
analysis of the
C170F and
C170W mutants by rapid reaction
kinetics similar to that previously conducted for wild type tryptophan
synthase (Anderson et al., 1991).
Figure SI:
Scheme I.
MATERIALS AND METHODS
Enzyme Purification
Tryptophan synthase



complex from S. typhimurium was isolated and purified as described (Miles et al., 1989). The
C170F and
C170W mutants of the



complex tryptophan synthase were
produced as described previously (Ruvinov et al., 1995) and
purified in a manner analogous to wild type.
Enzyme Assays
The activities of the



complex in Reactions 1 and 2 (Table 1) were measured by spectrophotometric assays at 37 °C
(Creighton, 1970). Reaction 3 was measured by a direct
spectrophotometric assay by monitoring the difference in absorption
between indole and L-tryptophan at 290 nm (Miles et
al., 1987). Reaction 4 was measured by a spectrophotometric assay
coupled with lactic dehydrogenase (Crawford and Ito, 1964). The
original assay for the activity of the
subunit was modified for
the 


complex by replacing ammonium
chloride with 0.18 M sodium chloride.
Chemicals
Unlabeled and
C-labeled IGP
were synthesized enzymatically using tryptophan synthase in the reverse
reaction with indole or [
C]indole and G3P
as described previously (Kawasaki et al., 1987). Radiolabeled
indole (specific activity 50 mCi/mM) was purchased from
Research Products International (Mount Prospect, IL). Glyceraldehyde
3-phosphate was prepared from the dimethyl acetal of glyceraldehyde
3-phosphate (Boehringer Manneheim) using the method of Racker et
al.(1959). Unlabeled standards of indole and tryptophan and serine
were purchased from Aldrich. All buffers and other reagents employed
were of highest commercial purity. Millipore ultrapure water was used
for all solutions. All experiments were conducted at 37 °C in
sodium Bicine buffer (50 mM) at pH 7.8
Stopped-flow Experiments
Stopped-flow measurements
were performed using a Kintek SF-2001 (Kintek Instruments, State
College, PA). This apparatus has a 1.5-ms dead time, a 0.5-cm path
length, and a thermostatted observation cell maintained at 37 °C.
The change in protein conformation upon serine binding was monitored by
following protein fluorescence using a monochromator set at 280 nm on
the input and monitoring the changes in intrinsic enzyme fluorescence
with an output filter at 340 nm. The serine-pyridoxal phosphate
complexes were examined by excitation at 405 nm and monitoring
fluorescence at 500 nm. In most experiments an average of four runs was
used for data analysis and a minimum of a 5-fold excess of the variable
substrate over enzyme was used to allow analysis as a pseudo-first
order rate constant. Rate constants were obtained by fitting the data
to a single or double exponential by non-linear regression.
Rapid Quench Experiments
The rapid quench
experiments were performed using a Kintek RFQ-3 Rapid-Chemical Quench
(Kintek Instruments). The reaction was initiated by mixing the enzyme
solution (15 µl) with the radiolabeled substrates (15 µl,
approximately 20,000 dpms). In all cases, the concentrations of enzyme
and substrates cited in the text are those after mixing and
during the enzymatic reaction. The reaction mixture was then quenched
by mixing with 67 µl of 0.6 M KOH to give a final
concentration of 0.2 M KOH during mixing in the rapid quench
apparatus. The quenched reaction solution was collected in a 1.5-ml
Eppendorf tube, vortexed, and analyzed by HPLC. The substrate and
products were quantified by HPLC as described below. In order to ensure
that the base was quenching the enzymatic reaction, a control was
included with each experiment to insure that catalysis was being
terminated. This involved adding the enzyme to a premixed solution of
base and substrates. Control experiments were also done to establish
the stability of the radiolabeled substrates under the quench
conditions employed.
HPLC Analysis
The substrates and products were
quantified by HPLC using a radioactivity detector. The HPLC separation
was performed using a BDS-Hypersil-C18 reverse phase column (250
4.6 mm, Keystone Scientific, Bellefonte, PA) with a flow rate
of 1 ml/min. The following gradient separation was employed where
solvent A is 0.2% sodium bicarbonate and solvent B is methanol. The
linear gradient program was as follows: 0-100% B in 20 min, hold
at 100% B for 10 min, recycle (100-0% B in 15 min), and then
re-equilibrate by holding at 100% A for 5 min. The elution times were
as follows: IGP, 7 min; tryptophan, 19 min; and indole, 23 min. The
HPLC effluent from the column was mixed with liquid scintillation
mixture (Mono-flow V, National Diagnostics) at a flow rate of 4 ml/min.
Radioactivity was monitored continuously using a Flo-One radioactivity
detector (Radiomatic Instruments, Tampa, FL).
Data Analysis
The KINSIM kinetic simulation
program (kindly provided by Carl Frieden and Bruce Barshop, Washington
University, St. Louis, MO; Barshop et al., 1983) was used to
model all of the kinetic data presented in this paper. The program was
modified to allow the input of data from the rapid quench experiments
as x,y pairs and to calculate the sum square errors in fitting
the data (Anderson et al., 1988). The entire data set was fit
to a single model and a single set of rate constants. The data were fit
by a trial and error process maintaining the constraints of known
dissociation constants and K
values for IGP, G3P,
indole, tryptophan, and serine (Nagata et al., 1989; Kawasaki et al., 1987; Phillips et al., 1984). Not all of the
rate constants are known with equal certainty. In the
reaction
the rates of IGP binding in the forward direction and of indole and G3P
binding in the reverse reaction are rough estimates based upon the
concentration dependence of the single turnover kinetics in each
direction. The corresponding dissociation rates for IGP, indole, and
G3P are then estimated from the approximate dissociation equilibrium
constants. Although these constants are not known with certainty, their
values do not greatly affect the fitting process or the interpretation.
The remaining rate constants involving the chemical interconversion of
IGP with indole and G3P were obtained as fits to the single turnover
kinetics in each direction and are therefore known with more certainty.
Regression analysis in the final stages of the fitting process place
the limits of error on the rate constants of approximately ±20%.
In the
reaction, the rates of serine reaction to form the
external aldimine (E-Ser) and the aminoacrylate (E
AA)
were obtained by direct fitting of stopped-flow fluorescence data as a
function of serine concentration and are within ±10%. The rates
of indole channeling and the reaction of indole with the aminoacrylate
to form tryptophan are set at the lower limit sufficient to account for
the accumulation of indole in the single turnover (Fig. 1, Fig. 3, and Fig. 6). The rate of tryptophan release is
determined by the presteady state burst experiments as well as the
steady state turnover rate analogous to previous analysis of wild type
tryptophan synthase. The reverse rate constants governing rebinding and
reaction of tryptophan are set according to K
and k
values for the reverse
reaction for wild
type tryptophan synthase (Weischet and Kirschner, 1976; Ahmed et
al., 1986), although they do not affect the interpretation of the
data presented in this paper.
Figure 1:
A single
turnover of
C170F mutant tryptophan synthase



complex in the 
reaction.
A solution of serine and [
C]IGP was mixed with
enzyme to initiate the reaction. The final concentrations of reactants
were as follows: 10 mM serine, 20 µM enzyme, and
8.77 µM [
C]IGP. The concentrations
shown refer to final concentrations after mixing. After various times,
the reaction was stopped by the addition of 0.2 M KOH and the
disappearance and formation of radiolabeled IGP (
), indole
(
), and tryptophan (
) were quantified by HPLC as described
under ``Materials and Methods.'' Each curve was calculated by
computer simulation of the kinetics using the rate constants summarized
in Fig. SII.
Figure 3:
Kinetics of a presteady state burst of the
C170F mutant tryptophan synthase



complex in the

reaction. A solution containing enzyme (20 µM)
and serine (10 mM) (pre-mixed) was added to a solution of
[
C]IGP (200 µM). The disappearance
and formation of IGP (
), indole (
), and tryptophan (
)
were monitored. The curves were calculated by the numerical integration
using the rate constants summarized in Fig. SII. B, the
curves were simulated using a rate of indole binding from solution of
0.03 µM
s
(0.6
s
at 20 µM enzyme). The rate of
reaction of indole from solution is too slow to account for the amount
of indole observed in the 
reaction
above.
Figure 6:
A single turnover of
C170W mutant
tryptophan synthase 


complex in the

reaction. A solution of serine and
[
C]IGP was mixed with enzyme to initiate the
reaction. The final concentrations were serine (100 mM),
enzyme (20 µM), and [
C]IGP (6
µM). The concentrations shown are final concentrations
after mixing. After various times, the reaction was stopped by the
addition of 0.2 M KOH and the disappearance and formation of
radiolabeled IGP (
), indole (
), and tryptophan (
)
were quantified by HPLC as described under ``Materials and
Methods.'' Each curve was calculated by computer simulation of the
kinetics using the rate constants summarized in Fig. SIII.
Figure SII:
Scheme II.
RESULTS
The rapid kinetic analysis described in this paper leads to
the pathways for the
C170F mutant and
C170W mutant shown in
Schemes II and III, respectively. Key rate constants obtained in
Schemes II and III are summarized in Table 2and compared with
those obtained for the wild type 


complex (Anderson et al., 1991). The curves shown in Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8were
calculated by numerical integration using one set of rate constants for
each mutant as summarized in Schemes II and III, with no simplifying
assumptions. The rates of indole channeling and the reaction of indole
with the aminoacrylate to form tryptophan are set at the lower limit
sufficient to account for the accumulation of indole in the single
turnover experiments. In fitting the data, the rate constants for the
reaction were further constrained by dissociation constants and K
values for IGP, G3P, serine, and tryptophan as
previously determined (Nagata et al., 1989; Kawasaki et
al., 1987; Phillips et al., 1984) and in Table 1in
this work.
Figure 2:
A single turnover
C170F mutant
tryptophan synthase 


complex in the
reaction. The
reaction of indole from solution. A solution
of serine and enzyme was mixed with a solution of
[
C]indole to initiate the reaction. The final
concentrations were serine (10 mM), enzyme (40
µM), and [
C]indole (6
µM). The disappearance and formation of indole (
)
and tryptophan (
) were monitored. B, the curves were
simulated using a rate of indole binding from solution of 0.03
µM
s
(0.6
s
at 20 µM enzyme). The rate of
reaction of indole from solution is too slow to account for the amount
of indole observed in the 
reaction
above.
Figure 4:
Kinetics of binding of serine of the
C170F mutant tryptophan synthase 


complex to the
subunit. A, a representative trace
from a stopped-flow experiment shows the time dependence of formation
and decay of the highly fluorescent serine-pyridoxal species termed the
external aldimine after mixing enzyme (2 µM) with serine
(5000 µM). Fluorescence excitation was at 405 nm and
emission was observed at 500 nm. The smooth line shows the fit to a
double exponential with rates for the fast and slow phases of 172
s
and 12.9 s
, respectively. B, the concentration dependence of the rate of serine reaction
for the fast phase. The rate of the faster phase was fit to a line
having a slope of 0.03 µM
s
which is the second-order rate constant for the
formation of the external aldimine species. C, the
concentration dependence of the rate of serine reaction for the slower
phase. The rates of the slower phase were fit to a hyperbola having a
maximum rate of 12.9 s
which corresponds to the rate
of formation of the aminoacrylate species.
Figure 5:
Protein conformational change upon serine
binding by the
C170F mutant tryptophan synthase



complex. A, a
representative trace from a stopped-flow experiment shows the time
dependence of a change in protein fluorescence after mixing enzyme (2
µM) with serine (2500 µM). The fluorescence
excitation was at 290 nm and emission was observed at 340 nm. The
smooth line shows the fit to a single exponential with a rate of 11.8
s
. B, the serine concentration dependence
of the rate of the change in protein fluorescence. The rates were fit
to a hyperbola having a maximum rate of 15
s
.
Figure 7:
A single turnover
C170W mutant
tryptophan synthase 


complex in the
reaction. The
reaction of indole from solution. A solution
of serine and enzyme was mixed with a solution of
[
C]indole to initiate the reaction. The final
concentrations were 100 mM serine, 40 µM enzyme,
and 2.4 µM [
C]indole. The
disappearance and formation of indole (
) and tryptophan (
)
were monitored. The curves were calculated by the numerical integration
using the rate constants summarized in Fig. SIII.
Figure 8:
Kinetics of a presteady state burst of the
C170W mutant tryptophan synthase



complex in the
reaction. A
solution containing enzyme (40 µM) and serine (100
mM) (pre-mixed) was added to a solution of
[
C]indole (200 µM). The
disappearance and formation of indole (
) and tryptophan (
)
were monitored. The curves were calculated by the numerical
integration using the rate constants summarized in Fig. SIII.
Steady State Kinetic Characterization of Mutants
A comparison of steady state kinetic parameters for wild type
and mutant tryptophan synthase is shown in Table 1. The steady
state enzyme turnover rates of the wild type and mutant



complexes are compared in Reactions
1-4. This characterization was important for providing direction
for an in-depth transient kinetic analysis. There were several
differences which are noteworthy of mention. First, the rate of the
reaction (Reaction 1) in the absence of serine for the
C170F
mutant is similar to that observed for wild type (0.16
s
) while the rate for the
C170W mutant is
2-fold slower (0.08 s
). Second, the K
for serine was similar for wild type and
C170F mutant (0.5
mM) while the K
for the
C170W mutant
was almost 100-fold higher (50 mM). Our previous analysis with
wild type tryptophan synthase has shown that the binding of serine
stimulates the rate of cleavage of IGP (
reaction). This
modulation of activity is mediated by an intersubunit communication in
which catalysis at the
subunit is triggered by the pyridoxal
phosphate activation of serine to form an aminoacrylate species and a
concomitant protein conformational change. This process in turn is
coupled to the production of indole at the
site and channeling to
the
site. The steady state rate of the 
reaction
(Reaction 2) is enhanced 30-fold for wild type (4.7
s
) and 7.5-fold for
C170F mutant (1.2
s
) in the presence of serine (Table 1). On the other hand, a less than 2-fold activation was
noted with the
C170W mutant (0.14 s
). The
slower rate of enhancement, particularly in the case of the
C170W
mutant, suggests that these mutants may be impaired in intersubunit
communication as well as channeling. The transient kinetic experiments
described below were designed to probe the channeling of indole and
intersubunit communication in the tryptophan synthase mutants. Finally,
in the absence of
site ligands (indole or IGP) tryptophan
synthase catalyzes a side reaction, the slow hydrolysis of serine to
form pyruvate, ammonia, and water (Table 1, Reaction 4). The
rates in Reaction 4 of the
C170F and
C170W mutants are 2- and
4-fold higher, respectively, than that of the wild type



tryptophan synthase. Since this side
reaction may complicate interpretation of results in experiments in
which serine is preincubated with enzyme, some of the single turnover
experiments were carried out by simultaneously mixing enzyme with a
solution of IGP and serine.
Kinetic Characterization of the
C170F Mutant
The rapid kinetic analysis of the
C170F mutant described
below leads to the kinetic pathway summarized in Fig. SIIand Table 2. The rate constants which distinguish wild type and
mutant are highlighted in boxes.
Kinetics of a Single Turnover: The 
Reaction
We have previously found that the examination of a
single enzyme turnover of the 
reaction is an important
diagnostic experiment to evaluate channel impairment. One of the
essential features of efficient channeling of indole is that the
transfer of indole from the
to the
subunit should be rapid
such that no indole should accumulate during a single enzyme
turnover. If passage of indole is blocked or impeded then it is likely
to be observed during this type of experiment. (In this experiment,
excess enzyme (20 µM) was mixed with a solution of serine
(10 mM) containing a limiting concentration of
[
C]IGP (8.7 µM), and the
disappearance of IGP and production [
C]indole
and tryptophan were monitored.) The observed production of indole (Fig. 1) provides the first kinetic evidence that the channeling
is impaired with the
C170F 


complex.
Kinetics of Reaction of Indole from Solution
In
order to establish that the indole is channeled from the
site to
the
site, we performed an experiment to assess the alternate
pathway involving dissociation of indole from the
site and fast
rebinding to the
site. The rate of reaction of indole from
solution was measured in a single turnover experiment using excess
enzyme (20 µM) preincubated with serine (10 mM).
The enzyme-serine complex was mixed with a limiting concentration of
[
C]indole (6 µM) and the production
of [
C]tryptophan was monitored. The
disappearance of indole and the formation of tryptophan are shown in Fig. 2. The rate of indole reaction from solution is
approximately 0.6 s
which is limited by the rate of
indole binding to the enzyme as confirmed by experiments performed at
different enzyme concentrations (data not shown). These data provided a
second-order rate constant of 0.03 µM
s
for the binding of indole to the enzyme. As shown
in Fig. 2B, the observed rate of reaction of indole
from solution (0.6 s
at 20 µM enzyme)
does not account for the amount of indole we observe in the 
single turnover.The combination of the two single turnover
experiments ( Fig. 1and Fig. 2) indicates that indole is
channeled from the
site to the
site in the physiologically
relevant 
reaction. Because the reaction of indole from
solution to form tryptophan is slow (0.6 s
) relative
to wild type (
40 s
), the indole observed during
the conversion of IGP to tryptophan in the 
reaction must
have been channeled otherwise a smaller amount of indole would have
accumulated in solution (see Fig. 2B). However, the
rate of channeling from the
to
site must have slowed from
>1000 s
in wild type to 100 s
in order to account for the observation of any indole. Therefore,
based upon this computer simulation of the reaction kinetics, we
conclude that indole must pass through the channel and it must do so
more slowly than in the wild type enzyme.
Kinetics of the 
Presteady State Burst
Reaction
We sought to provide additional information which would
discern whether the indole observed in the single turnover experiment
was actually trapped in the tunnel or free in solution. Molecular
modeling studies based upon the tryptophan synthase crystal structure
have suggested that the hydrophobic channel could accommodate up to 4
molecules of indole per enzyme site (Hyde et al., 1988).
Therefore we examined the 
reaction in a presteady state
burst experiment in which substrate is in slight excess over enzyme, to
determine whether indole which is trapped in the tunnel builds up to a
constant level (Fig. 3).The time course for the forward
reaction was determined by mixing a solution containing enzyme and
serine (pre-mixed) with a solution containing
[
C]IGP to initiate the reaction. The final
concentrations after mixing were 20 µM enzyme, 10 mM serine, and 200 µM [
C]IGP. At
various times after mixing, the reaction was quenched by the addition
of 0.2 M KOH (final concentration) and the quenched reaction
mixture was then analyzed by HPLC as described under ``Materials
Methods.'' The time dependent disappearance of IGP and formation
of radiolabeled indole and tryptophan are shown in Fig. 3. The
results show a presteady burst of product formation with an amplitude
of 0.2 per enzyme site and a rate of 30 s
which was
followed by a slower rate of 3 s
corresponding to
steady state turnover. As anticipated, we observed the accumulation of
a relatively constant quantity of indole corresponding to concentration
of approximately 15-20 µM or
1 indole per
enzyme site. If the indole formed was in solution, a very slow
accumulation of indole with a lag would be predicted (see Fig. 3B) and as illustrated does not fit the
experimental data. Thus this experiment provides further support for
the suggestion that the observed indole is in the tunnel rather than in
solution.
Kinetics of the
Reaction
Our previous
examination of the conversion of IGP to indole in a single turnover
(
reaction) in the absence of serine with wild type tryptophan
synthase showed that there is not a presteady state burst of indole
formation. This indicated that the slow rate of IGP cleavage in the
absence of serine is not the result of indole that is held very tightly
at the active site of the
subunit and released into solution very
slowly (Anderson et al., 1991). Similar results were obtained
with the
C170F mutant (data not shown) as no burst of indole
formation is observed; rather the very slow formation of indole occurs
at the steady state rate of (0.16 s
). Since
chemistry is not rate-limiting (see below), this experiment suggests
that a step preceding chemistry is rate-limiting. Repeating the
experiment at a higher enzyme concentration did not give an increase in
rate, indicating that the initial binding rate for IGP was not
rate-limiting. In addition, the observed rate was equal to the steady
state rate at saturating IGP concentration. Taken together, these data
suggest that there is a rate-limiting conformational change which must
occur after the initial binding of IGP and limits the rate of the
chemical reaction. In the presence of serine this conformational change
must occur much faster. These results are very similar to those
previously obtained with wild type tryptophan synthase. The rate of the
chemical reaction observed here is consistent with the rate of IGP
cleavage observed in the forward 
reaction (with serine),
suggesting that only the rate of the conformational change which limits
the
reaction (IGP-E
IGP-E*) is affected
by the presence of serine.
Serine Modulates the
Reaction
In the kinetic
pathway of wild type tryptophan synthase, it has been shown that a
protein conformational change occurs prior to and limits the rate of
IGP cleavage. The rate of tryptophan formation (in the presence of
serine) is much faster than the cleavage of IGP in the absence of
serine, since serine increases the rate of this protein conformational
change occurring at the
subunit. According to this model, the
enzyme is converted from a less active state to a more active state
upon reaction with serine at the
site. Support for this model was
provided in earlier studies with wild type enzyme by comparison of a
single turnover 
reaction in which serine is preincubated
with enzyme versus the simultaneous addition of IGP and serine
to enzyme. A lag in tryptophan production was observed in the
experiment in which there was no preincubation, indicating
that IGP cleavage at the
site is slow until serine binds to the
site. A similar comparison could not be made with the
C170F
mutant since the faster rate of serine hydrolysis complicated the

single turnover experiment in which enzyme is preincubated
with serine. Nonetheless, this serine activation step appears to be
present in the kinetic pathway of the
C170F mutant as evidenced by
the slow rate of IGP cleavage in the absence of serine and the much
faster IGP cleavage during the single turnover of the 
reaction in the presence of serine. Our estimated rate of IGP cleavage
in the presence of serine (24 s
in Fig. SIIand Table 2) is 150-fold higher than the rate in
the absence of serine (0.16 s
in Fig. SIIand Table 2). This 150-fold activation by serine is similar to that
observed with the wild type enzyme (Anderson et al.(1991) and Table 2). The magnitude of this activation step is masked in the
steady state comparison since product release is the rate-limiting
step.
Kinetics of Serine Reaction Measured by Stopped-flow
Fluorescence Methods
To further define the activation step we
examined the kinetics of serine binding. It has previously been shown
that there are absorbance and fluorescence changes exhibited by the
pyridoxal phosphate upon reaction with serine and chemical nature of
the serine-pyridoxal phosphate complexes have been defined (Faeder and
Hammes, 1971; York, 1972; Lane and Kirschner, 1983; Lane et
al., 1984; Drewe and Dunn, 1985). In the first step, serine
displaces the internal aldimine formed between Lys-87 and the pyridoxal
phosphate at the active site of the
subunit to form the external
aldimine (E-Ser in Schemes II and III), which is highly fluorescent,
with emission at 500 nm upon excitation at 405 nm. The
proton of
serine is then removed to form a quinonoid species which is
subsequently dehydrated to form a reactive aminoacrylate (E
AA in Schemes II and III). The electrophilic aminoacrylate
is then poised to react with the incoming nucleophile, indole, in the
physiological reaction to form tryptophan. If no indole is present the
aminoacrylate is slowly hydrolyzed to form pyridoxal phosphate,
pyruvate, and ammonia (Reaction 4 in Table 1).In the current
study, we focused on measuring the rate of formation of the external
aldimine and of its decay to the aminoacrylate by stopped-flow
fluorescence because of the increased sensitivity of these methods. A
representative fluorescence trace is shown in Fig. 4A.
The data were fit to a double exponential to measure the rates of
formation and decay of the external aldimine. The dependence of the
rates of the fast and slow phases on serine concentration are shown in Fig. 4, B and C. The data for the faster phase
(formation of the external aldimine) were fit to a straight line giving
a second-order rate constant of 0.03 µM
s
and showed no signs of leveling off at the higher
serine concentrations (Fig. 4B). The concentration
dependence of the rate of the slower phase (decay to the aminoacrylate)
was fit to a hyperbola which reached an observed maximum with a rate of
12 s
. Complete solution for the two-step reaction
kinetics (Johnson, 1992) yielded all four rate constants for the
reaction as shown below and summarized in Fig. SII:

On-line formulae not verified for accuracy
with k
= 0.03
µM
s
, k
= 31 s
, k
= 5 s
, and k
= 7 s
. Thus the
rate of aminoacrylate formation for the
C170F mutant (12
s
) is approximately 4-fold slower than wild type (45
s
, Table 2).
Changes in Protein Conformation Upon Serine
Binding
Our earlier studies have indicated that the activation
at the
site is mediated by a change in protein conformation. A
change in intrinsic protein fluorescence was observed when wild type
tryptophan synthase was mixed with serine. It was found that the rate
of the protein conformational change was coincident with the rate of
formation of aminoacrylate. These data suggest that the formation of
the aminoacrylate at the
site elicits a protein conformational
change which is transmitted to the
site where it triggers the
cleavage of IGP to indole and G3P (Anderson et al., 1991).
Similar results were obtained with the
C170F mutant. A
representative trace illustrating the protein conformation is shown in Fig. 5A. The concentration dependence of the rate of
the fluorescence change (Fig. 5B) was fit to a
hyperbola giving a maximum rate of 15 s
. In
conclusion, the kinetics of serine binding occur at a slower rate than
wild type. The rate of the aminoacrylate formation (12
s
) is accompanied by a protein conformational change
occurring at a similar rate (15 s
).
Kinetic Characterization of the
C170W Mutant
The effects on efficient channeling of indole proved much
more pronounced with the
C170W mutation. Earlier studies on the
C170W mutant provided kinetic and structural evidence that the
channel was impaired (Schlichting et al., 1994). The steady
state kinetic data as well as the transient kinetic data presented
below indicate that the mutation has not only blocked or impeded the
passage of indole from the
to
site but also has affected

intersubunit communication. The rapid kinetic analysis of
the
C170W mutant described below leads to the kinetic pathway
summarized in Fig. SIII and Table 2. This in-depth
analysis provided results in reasonable agreement with a previous
preliminary report (Schlichting et al., 1994). The rate
constants which distinguish the wild type from
C170W are
highlighted in boxes.
Kinetics of a Single Turnover: The 
Reaction
A
single enzyme turnover of the 
reaction for the
C170W
mutant is shown in Fig. 6. In this experiment, excess enzyme (20
µM) was mixed with a solution of serine (100 mM)
containing a limiting concentration of [
C]IGP (6
µM), and the disappearance of IGP and production
[
C]indole and tryptophan were monitored. As
shown in the figure a small amount of indole is observed during the
conversion of substrate to product. The rate of IGP cleavage to indole
is similar in the presence (0.12 s
) or absence (0.08
s
) of serine. When the experiment was repeated at
higher enzyme concentration there was no change in the rate of indole
or tryptophan formation indicating that the binding of IGP is not
limiting the reaction. Since the rate of IGP cleavage is so slow and
there is little or no activation of the
reaction by the presence
of serine, we do not know if the indole we observe is in solution or
trapped in the tunnel.
Kinetics of Reaction of Indole from Solution
The
rate of reaction of indole from solution was measured in a single
turnover experiment using excess enzyme (20 µM)
preincubated with serine (100 mM). The enzyme-serine complex
was mixed with a limiting concentration of
[
C]indole (2.4 µM) and the
production of [
C]tryptophan was monitored. The
disappearance of indole and the formation of tryptophan are shown in Fig. 7. A comparison of the time scale for Fig. 6and Fig. 7shows that the
reaction is at least 20-fold faster.
The observed rate of indole reaction from solution is approximately
0.35 s
. The rate of the
single turnover
reaction is limited by the rate of indole binding to the enzyme as
confirmed by experiments performed at different enzyme concentrations
(data not shown). These data provided a second-order rate constant of
0.03 µM
s
for the
binding of indole to the enzyme. This was similar to the rate
determined for the
C170F mutant and approximately 60-fold slower
than wild type (Table 2). Since the rate of reaction of indole
from solution is similar to the rate of IGP cleavage in the single
turnover experiment (0.35 s
versus 0.12
s
) it is difficult to distinguish whether the indole
we observed is in solution or is trapped in the tunnel. However,
computer simulations suggest that if the channeling step is eliminated
no indole would be observed. Therefore, the indole we observe in the
single turnover experiment may actually be trapped in the tunnel.
Kinetics of the
Presteady State Burst
Reaction
A presteady state burst experiment to examine the

reaction for the
C170W mutant will not be informative
since the reaction will be limited by the rate of IGP cleavage to
indole and therefore no burst of product will be observed. On the other
hand, a presteady state analysis of the
reaction did show a burst
of product formation. The time course for the forward reaction was
determined by mixing a solution containing enzyme and serine
(pre-mixed) with a solution containing [
C]indole
to initiate the reaction. The final concentrations after mixing were 40
µM enzyme, 100 mM serine, and 200 µM [
C]indole. At various times after mixing,
the reaction was quenched by the addition of 0.2 M KOH (final
concentration) and the quenched reaction mixture was then analyzed by
HPLC as described under ``Materials and Methods.'' The time
dependent disappearance of indole and formation of radiolabeled
tryptophan are shown in Fig. 8. A presteady burst of product
formation was observed with an amplitude of 0.4 per enzyme site and a
rate of 6 s
(indole binding rate-limiting) followed
by a slower rate of 1 s
corresponding to steady
state turnover.
Kinetics of the
Reaction
The examination of
the
reaction for the
C170W mutant revealed a slow rate of
IGP cleavage (0.08 s
) similar to
C170F and wild
type (data not shown). As anticipated there was no burst of product
indole formation and the rate did not increase with higher enzyme
concentration indicating the binding to the enzyme was not
rate-limiting. Again, these data indicate that there is a rate-limiting
conformational change which must occur after the initial binding of IGP
and limits the rate of the chemical reaction.
Serine Does Not Modulate the
Reaction
One of
the most surprising results with the
C170W mutant was the lack of
substantial activation of the
reaction in the presence of serine,
especially since the
C170F mutant behaved very similar to wild
type. In order to understand this lack of stimulation by serine we
wanted to take a closer look at the interaction of serine with the
enzyme. There are several possibilities which may have lead to the
observed results. First the aminoacrylate formation may be altered
since it is this chemical species which is involved in the activation
step. Second, there may be a problem with 
intersubunit
communication due to an alteration in the protein conformational
change. Finally, although the
C170W is remote from the
active site, it could nonetheless be important in the triggering step.
This possibility might be less likely since the
C170F does not
exhibit the same type impairment.
Kinetics of Serine Reaction Measured by Stopped-flow
Fluorescence Methods
The rate of formation of the external
aldimine and its decay to the aminoacrylate were measured by
stopped-flow fluorescence as described above. We could not observe the
aminoacrylate at lower concentrations of serine. However, at higher
concentrations of serine (close to the K
) the
decay of the external aldimine to the aminoacrylate was observed
similar to that observed for the
C107F mutant (data not shown).
The data over a range of serine concentrations were fit to a double
exponential to measure the rates of formation and decay of the external
aldimine. The dependence of the rates of the fast and slow phases on
serine concentration was determined. The data for the faster phase
(formation of the external aldimine) were fit to a straight line giving
a second-order rate constant of 0.002
µM
s
and showed no
sign of leveling off at the higher serine concentrations. The
concentration dependence of the rate of the slower phase (decay to the
aminoacrylate) was fit to a hyperbola which reached an observed maximum
with a rate of 12 s
. Complete solution for the
two-step reaction kinetics (Johnson, 1992) yielded all four rate
constants for the reaction: with k
= 0.002
µM
s
, k
= 156 s
, k
= 9 s
, and k
= 3 s
as
summarized in Fig. SIII. These results indicate that the
equilibrium between external aldimine and aminoacrylate is
substantially changed such that a very high concentration of serine is
required to shift the equilibrium toward aminoacrylate.
Changes in Protein Conformation Upon Serine
Binding
When we examined the intrinsic protein fluorescence upon
binding serine, a protein conformational change was not observed even
at higher serine concentrations. It is possible that the protein
conformational change necessary to activate the
reaction has now
become uncoupled from the formation of the aminoacrylate. An alternate
explanation involves interference or fluorescence quenching due to the
proximity between the
170 and
177 tryptophans. The
177
tryptophan is located approximately 7 Å from
170 tryptophan (
)and thus may interfere with the detection of a change in
protein fluorescence although similar effects might have been expected
to occur with the phenylalanine of the
C170F mutant.
DISCUSSION
The use of rapid chemical quench-flow methods to provide a
direct kinetic analysis of mutant forms of tryptophan synthase has
allowed comparison with wild type and has given us an opportunity to
test our current model of channeling and the kinetic consequences of
the channel mutations. Several surprising results were obtained related
to substrate channeling and protein communication.
According to our
model for the kinetics of efficient channeling of indole, we have
established as predicted that indole can be observed in a single enzyme
turnover of the 
reaction if the rate of passage of indole
and/or the rate of chemistry at the
subunit are decreased
(Schemes II and III and Table 2). The phenylalanine mutation
slows the rate of channeling approximately 10-fold compared with wild
type, whereas the tryptophan mutation reduces the rate >1000-fold.
Although this difference in the two mutations may be related to the
size of the obstructing residue, it could also be related indirectly to
the lack of activation of the
reaction (Fig. SIII and Table 2) by the aminoacrylate in the
C170W mutation. We
speculate that the activation of IGP cleavage at the
site is
prevented by the presence of the indole ring of
Trp-170 which may
mimic the indole intermediate in the tunnel. These results are
supported by our observations that a protein conformational change in
intrinsic protein fluorescence is not detected with this mutant. In
addition, preliminary structural analysis indicates that the
C170W
mutation not only restricts the tunnel but also results in the movement
of residues which may be important in transmitting the activation or
trigger from the
subunit to the
subunit.
For
instance, in the presence of 5-fluoroindole propanol phosphate (an
inhibitor of the
reaction) there is a 10-Å movement of the
guanidinium group of
Arg-175 from the surface of the protein in
the native wild type tryptophan synthase without ligand to the

interface in the
C170W mutant structure. The
combination of kinetic results presented here as well as more complete
structural analysis of this mutant may provide important clues
concerning 
intersubunit communication.
In summary, the
kinetic analysis of the mutant enzymes presented in this report
reaffirms the essential kinetic features required for efficient
channeling. Slowing the rate of passage of indole and/or the rate of
reaction of indole at the
site (Anderson et al., 1991)
leads to the detection of the indole in a single enzyme turnover of the
physiological reaction. Even though the
C170F mutant intersubunit
communication maintains coupling of the
and
reactions, the
efficiency of channeling is decreased. Efforts to define the structural
basis for this conformational coupling by x-ray crystallography,
solid-state nmr, and site-directed mutagenesis are currently underway.