(Received for publication, November 22, 1994; and in revised form, January 12, 1995)
From the
The bacterial tryptophan synthase complex contains an unusual structural feature: an intramolecular
tunnel that channels indole from the active site of the
subunit
to the active site of the
subunit 25 Å away. Here we
investigate the role of the tunnel in communication between the
and
subunits using the polarity-sensitive fluorescent probe, Nile
Red. Interaction of Nile Red in the nonpolar tunnel near
subunit
residues Cys-170 and Phe-280 is supported by studies with enzymes
altered at these positions. Restricting the tunnel by enlarging Cys-170
by chemical modification or mutagenesis decreases the fluorescence of
Nile Red by 30-70%. Removal of a partial restriction in the
tunnel by replacing Phe-280 by Cys or Ser increases the fluorescence of
Nile Red more than 2-fold. A binding site for Nile Red in this region
near the pyridoxal phosphate coenzyme of the
subunit is further
supported by iodide quenching and fluorescence energy transfer
experiments and by molecular modeling based on the three-dimensional
structure of the
complex. Finally,
studies using Nile Red as a sensitive probe of conformational changes
in the tunnel reveal that allosteric ligands (
subunit) or active
site ligands (
subunit) decrease the fluorescence of Nile Red. We
speculate that allosteric and active site ligands induce a tunnel
restriction near Phe-280 that serves as a gate to control passage of
indole through the tunnel.
Metabolite channeling, the subject of considerable recent
interest and some controversy(1, 2) , is the process
by which the product of an enzymatic reaction is passed directly to the
next enzyme in a biosynthetic pathway rather than by diffusion through
solution. Tryptophan synthase (EC 4.2.1.20), a classic enzyme that
exhibits metabolite channeling, catalyzes the last two steps in the
biosynthesis of L-tryptophan. The bacterial enzyme is an
complex that dissociates reversibly
into monomeric
subunits and a
dimer (for
reviews, see (3, 4, 5, 6, 7) ). The
subunit catalyzes cleavage of indole-3-glycerol phosphate to
indole and D-glyceraldehyde 3-phosphate, termed the
reaction. The
subunit catalyzes the pyridoxal phosphate-dependent
condensation of indole with L-serine to form L-tryptophan, termed the
reaction. The physiologically
important reaction catalyzed by the
complex, termed the
reaction, is the sum of the
and
reactions (Fig. SIA). The finding that indole
does not appear as a free intermediate in solution in the
reaction provides evidence that indole passes intramolecularly from the
site to the
site(8, 9, 10) .
Channeling of indole is supported by rapid kinetic
experiments(11, 12, 13, 14) . The
crystal structure of the tryptophan synthase
complex from Salmonella
typhimurium(15) has provided a plausible structural basis
for channeling by revealing a
25-Å tunnel that connects the
catalytic sites of the
and
subunits.
Scheme I:
A, in
the tryptophan synthase reaction indole-3-glycerol phosphate (IGP) is cleaved to D-glyceraldehyde 3-phosphate (G-3-P) and an indole intermediate ([IND])
at the
site. The indole intermediate diffuses through a
25-30 Å tunnel to the
site where it undergoes a
pyridoxal phosphate (PLP)-dependent reaction with L-Ser to form L-Trp. B, active site of the
subunit based on the x-ray crystallographic results(15) .
The figure, which was prepared by C. C, Hyde, shows cysteine 170,
phenylalanine 280, the pyridoxal phosphate:Lys-87 Schiff base, indole
positioned near the end of a tunnel that extends from the
site to
the
site, and the approximate surface of the tunnel (dots). The dashed side chains show the positions of
phenylalanine 170 created by computer graphics modeling and an
alternative position of phenylalanine 280 seen in some crystal
structures (see text). The surface of the tunnel, shown by dots, has been described(15) . The
/
interaction site is located to the left of region shown. This
site and pyridoxal phosphate are approximately equidistant from
Cys-170.
Fig. SIB shows part of the tunnel within the subunit, the Schiff base
formed between pyridoxal phosphate and the
-amino group of lysine
87 at the active site of the
subunit, and two residues in the
wall of the tunnel, Cys-170 and Phe-280. Molecular modeling suggests
that a bulky side chain at position 170 might restrict the tunnel and
interact with the aromatic side chain of Phe-280, a residue on the
opposite wall of the tunnel that appears to enter the tunnel in some
crystal structures. (
)The alternative position of the chain
of Phe-280 is shown by a dashed line in Fig. SIB. Furthermore, substitution of Phe-280 by a
less bulky residue might prevent restriction of the tunnel at this
position. Fig. SIB also shows an indole molecule
positioned by computer graphics modeling at a likely position for
reaction with the L-serine-pyridoxal phosphate intermediate to
form L-tryptophan.
In the present work, we have followed
the suggestions of molecular modeling described above to construct
several specifically altered forms of the complex. To increase the bulk of
subunit Cys-170, we have
used chemical modification by N-ethylmaleimide to prepare
C170-NEM (
)and by methyl methanethiolsulfonate to make
C170-MMTS. We have also used site-directed mutagenesis to replace
Cys-170 by phenylalanine (C170F) and by tryptophan (C170W). Recent
crystallographic and kinetic analyses of the C170W mutant form of the
complex prepared as described here
for the first time show that the engineered tryptophan side chain does
indeed obstruct the tunnel and results in the transient accumulation of
an indole intermediate(16) . To reduce the partial restriction
of the tunnel by Phe-280, we have replaced Phe-280 by cysteine (F280C)
and by serine (F280S). The several new forms of the
complex engineered in the present
work should provide useful materials for future studies of the kinetics
of indole channeling.
Because residues that form the surface of the
tunnel are largely nonpolar(15) , nonpolar compounds other than
indole may bind in the tunnel and serve as probes of tunnel structure
and function. In the present work, we investigate the interaction of
the polarity-sensitive fluorescent dye, Nile
Red(17, 18, 19) , with various forms of wild
type tryptophan synthase and with complexes having alterations in the tunnel. Our results provide
evidence that Nile Red binds in the tunnel near the active site of the
subunit and that the geometry of this region of the tunnel is
altered by
site ligands and by allosteric
site ligands.
These ligands may induce a tunnel restriction near Phe-280 that serves
as a gate to control passage of indole through the tunnel.
R is the Forster critical distance at which
transfer of excitation energy is 50%. R
is defined
(in Angstroms) by,
where n is the refractive index, taken as 1.4,
is the donor quantum yield, taken to be 0.018 for
pyridoxal phosphate (40) and 0.027 for reduced pyridoxal
phosphate,
is an orientation factor that can have
values between 0 and 4 and is two-thirds if the fluorophores can freely
rotate, and J
is the spectral overlap integral. J
was evaluated numerically using 10-nm interval
values from the normalized emission spectra of pyridoxal phosphate (or
reduced pyridoxal phosphate) and the absorbance spectrum of Nile Red in
80% dioxane/water, a solvent in which Nile Red emission has properties
close to those of the enzyme-bound dye.
The efficiency of excitation transfer from donor to acceptor, E, is defined as follows,
where F is the fluorescence of the donor in
the absence of acceptor and F
is the
corresponding donor fluorescence in the presence of saturating
acceptor. Since the solubility of Nile Red in aqueous solution
prevented achievement of saturation, the observed transfer efficiency
must be corrected for the fraction bound. This was done by
extrapolation. Emission spectra of pyridoxal phosphate or reduced
pyridoxal phosphate were recorded in the absence of Nile Red and in the
presence of varying Nile Red concentrations up to 1 µM,
corresponding to a free dye concentration of about 0.2 µM based on the determined K
of
1
µM. The reduction in integrated intensity
(
F) of emission was determined for each concentration of
Nile Red. The value of
F at saturation was estimated from
the intercept of the data plotted in double-reciprocal format (42; see
also (43) ),
where B is a constant and
[L] is the concentration of free ligand,
here Nile Red.
The evaluation of spectral overlap integrals yielded
an overlap integral value of J = 19
10
M
cm
nm
for pyridoxal phosphate and Nile Red and a value
of J
= 1.1
10
M
cm
nm
for reduced pyridoxal phosphate and Nile Red. These values
correspond to critical Forster distances (R
) of 28
and 19 Å, respectively, assuming rotational averaging of the
fluorophores' emission. Orientation effects may not be trivial,
as steady-state and time-resolved measurements indicate significant
anisotropy for the Nile Red with the
complex compared with Nile Red in solution (data not shown). If
the assumption of rotational averaging in the donor-acceptor pair is
not valid, the value of R
for the reduced
pyridoxal phosphate-Nile Red pair might have the range of 8-22
Å.
To define functional roles of the tunnel that connects the
active sites of tryptophan synthase and
subunits (Fig. SI), we have engineered alterations in
subunit
residues Cys-170 and Phe-280 by site-directed mutagenesis and chemical
modification (see the Introduction and ``Methods and
Materials''). We have compared the interaction of the wild type
and altered forms of the tryptophan synthase
complex with the polarity sensitive
dye Nile Red to obtain evidence for its site of interaction. Finally,
we have analyzed the effects of active site (
subunit) and
allosteric ligands (
subunit) on the interaction of Nile Red with
the
complex.
Figure 1:
Emission spectra of Nile Red in the
presence of the tryptophan synthase and holo-
subunits and
holo-
complex. The structure of Nile
Red is given in A. Proteins (5 µM protomer in
Buffer B) were mixed with Nile Red (1 µM). Spectra were
taken after 15 min at room temperature and were deconvoluted as
described under ``Material and Methods.'' A,
subunit; B, holo-
subunit; C,
holo-
. Each panel shows the total
emission and the components revealed by deconvolution. The aqueous
component is characterized by higher wavelength emission. The
non-aqueous component emits at lower wavelength and is referred to in
the text and Table 1and Table 3as the ``nonpolar
component.'' Note that the total intensities and nonpolar
components of curves for the holo-
subunits in B are similar to
the intensities of the corresponding curves for the
holo-
complex in C. Details
of the total and nonpolar component emission are given in Table 1for different forms of the
enzyme.
Figure 2:
Emission spectra of Nile Red in the
presence of wild type and altered forms of the tryptophan synthase
holo- complex. A, wild type
holo-
complex and
holo-
complexes with modifications at
subunit Cys-170 (C170-MMTS, C170-NEM, C170F, and C170W). B, wild type
holo-
complex and
holo-
complexes with modifications at
subunit Phe-280 (F280S and F280C). Details of the total emission
and nonpolar components are given in Table 1for different forms
of the enzyme.
Figure 3:
Titration of the
holo- complex with Nile Red. A, holo-
complex (5
µM) was treated with Nile Red (0.2 to 2 µM)
for 15 min before recording of the emission spectra. B, curves
from A with 0.2 and 2 µM Nile Red are normalized to the
same maximum intensity. C, the reciprocal integrated emission
intensity of the nonpolar component derived from the deconvolution of
each curve in A and analogous curves collected in the presence
of ligands is plotted versus the reciprocal concentration of
Nile Red.
, no ligand;
, 4 mM indole;
, 1
mMD-tryptophan.
Figure 4:
Iodide quenching of the fluorescence of
pyridoxal phosphate (A) and Nile Red (B) bound to the
wild type, F280C, and F280S holo- complexes. See ``Materials and Methods.''
, wild
type;
, F280C;
, F280S.
Table 2presents the results of iodide
quenching of Nile Red bound to the wild type holo- subunit and to
the wild type and F280S and F280C holo-
complexes. Emission from Nile Red bound to the
subunit
could not be evaluated, since the presence of even 50-100 mM KI apparently destabilized the protein and altered the Nile Red
spectrum (data not shown). However,
subunit that was cleaved at
Arg-188 by trypsin (51) (``nicked''
subunit;
see ``Materials and Methods'') was not destabilized by KI.
Thus iodide quenching results with the nicked
subunit are
presented in Table 2. Fluorescence of Nile Red bound to nicked
subunit was poorly quenched by KI, exhibiting a Stern-Volmer
constant (K
) of <1 M
. It is noteworthy that the fluorescence
of indole-3-propanol phosphate bound to the
subunit is also
poorly quenched (by acrylamide)(40) . KI did quench Nile Red
emission from the holo-
subunit or
holo-
complex without destabilizing
these enzymes. The K
values obtained with the
holo-
subunit and holo-
complex
were similar and were larger than that obtained with the nicked
subunit, supporting the view that Nile Red is bound to
in the
complex.
The K values for Nile Red obtained with the F280C and F280S
complexes were much higher than that
for the wild type holo-
complex (Table 2). Comparison with quenching of pyridoxal phosphate
showed that quenching constants for both Nile Red and pyridoxal
phosphate were in the order: wild type < F280C
F280S ( Table 2and Fig. 4), indicating that the mutations affected
the environments of the pyridoxal phosphate and Nile Red similarly,
consistent with the suggestion that the two fluorophores are close
together.
To determine the mechanism of KI quenching, we obtained
fluorescence lifetimes of the Nile Red- complex in the absence or presence of KI. Surprisingly, the
lifetimes were nearly unchanged by sufficient KI to yield 30-50%
quench (data not shown). This was true for the wild type enzyme and for
the F280S and F280C enzymes, for which the K
values were much higher. Thus in all cases, the mechanism of
quenching is static, not collisional, since this would result in
parallel decrease in intensity and lifetime, as observed in
dimethylformamide.
Since the quenching is not collisional, the
extent of shielding of the bound Nile Red from free solvent cannot be
evaluated quantitatively. ()However, the results do indicate
that the bound Nile Red is protected from free contact with KI, since
the quenching in dimethylformamide proves that iodide is an efficient
collisonal quencher, if it can contact the Nile Red. In addition, the
similar responses of KI quenching of Nile Red and pyridoxal phosphate
to alteration of the Phe-280 residue suggest proximity of the
fluorophores.
The data in Fig. 5demonstrate transfer of
excitation energy from reduced-pyridoxal phosphate to Nile Red in the
wild type complex by showing
decreased reduced pyridoxal phosphate emission with increasing
concentrations of Nile Red. Because the concentrations of Nile Red in Fig. 5are not saturating, the decrease in reduced-pyridoxal
phosphate fluorescence at saturation must be calculated, as shown in
the inset and detailed under ``Materials and
Methods.'' Similar analysis was performed with the reduced and
holoenzyme forms of the wild type, F280C, and F280S
complexes. Significant energy
transfer was found in all cases, with extrapolated efficiencies of
transfer in the range of 0.55-0.85 for wild type and F280C
complexes. Transfer efficiencies
were higher with the holo-
complexes
than with the reduced
complexes,
although the error ranges overlapped. These values for transfer
efficiency indicate a separation distance of about 0.75-0.97
times the critical Förster distance, R
, corresponding to 14-27 Å. This
result is consistent with Nile Red binding near Cys-170, which is about
16 Å from pyridoxal phosphate (see Fig. SIB).
Figure 5:
Energy transfer from excited reduced
pyridoxal phosphate to Nile Red. Reduced pyridoxal phosphate
fluorescence emission from 5 µM reduced
complex was recorded upon direct
excitation at 315 nm with 0, 0.4, 0.6, 0.8, and 1.0 µM Nile Red added (top to bottom). The data clearly
show the progressive reduction in intensity of pyridoxal fluorescence
with increased Nile Red addition. The inset shows the
reduction in integrated fluorescence (
F) plotted versus the free Nile Red concentration (calculated as
described under ``Materials and Methods''), with the data
presented in double-reciprocal format. The reciprocal of the y intercept gives the value of
F at saturation, which
yields the value of E, the efficiency of energy transfer at
saturation. Here the data indicate that at saturation with Nile Red,
the pyridoxal fluorescence would be decreased by 76% or E = 0.76. The experiment was also performed with the
holo-
complex (excitation at 410 nm)
and with the holoenzyme and reduced forms of the F280S and F280C
complex (data not
shown).
Figure 6:
Computer graphics modeling of Nile Red in
the tryptophan synthase complex. The
model is based on the x-ray crystallographic results (15) and
K. D. Parris, C. C. Hyde, S. A. Ahmed, E. W. Miles, and D. R. Davies,
unpublished results. The side chains of Phe-280 and Tyr-279 have been
removed to show the open tunnel conformation described under
``Results.'' Close up of the
site showing the pyridoxal
phosphate:Lys-87 Schiff base (PLP), the approximate surface of
the tunnel (dots) nearby and Cys-170 (C-170). The
Nile Red molecule (NR) (
12 Å long, 6 Å wide,
and 3.4 Å thick) was generated by computer graphics modeling and
positioned in the optimal position in the tunnel (
16 Å long,
9 Å wide, and 4.5 Å deep in this
region).
The position of
Nile Red directly adjacent to Cys-170 is clearly consistent with the
Cys-170 and Phe-280 modification and substitution results, as well as
with the energy transfer results. This site should be available in the
F280C and F280S complexes because
the large phenylalanine that enters the tunnel in this location has
been replaced by a smaller residue. This may be available some of the
time in the wild type complex if an equilibrium occurs between open and
closed conformations (see ``Discussion'').
Using the
closed tunnel model, Nile Red could only be fit in the site in a
somewhat more restricted site closer to the pyridoxal phosphate in a
position that overlaps the site which tryptophan occupies when the
pyridoxal phosphate-L-tryptophan Schiff base is present in the
subunit active site. (
)In this position, the center of
the pyridoxal phosphate ring is 3.5 Å from the nearest Nile Red
ring center and about 11 Å from the nitrogen of Nile Red. Nile
Red is quite restricted in possible motions in the absence of movements
in the protein matrix. Rotation about either the long or short axis of
the molecule is very restricted and movement into the tunnel or through
the tunnel is not possible. A nonpolar area large enough to contain
Nile Red could not be identified either on the surface of the
complex or at the
interface. One site that could accommodate Nile Red was found in the
active site of the
subunit near the beginning of the tunnel.
Figure 7:
Effects of ligands on the emission spectra
of Nile Red with the subunit (A), the
subunit (B), and the wild type holo-
complex (C). Conditions are the same as in Fig. 1. Enzyme alone (curve 1), in the presence of 80
mMDL-
-glycerol 3-phosphate (curve 2),
in the presence of 50 mML-serine (curve 3),
or in the presence of both ligands (curve 4). The order of
addition of Nile Red and ligands did not affect the final spectra
obtained.
Figure 8:
Effect of L-serine concentration
on the fluorescence emission of Nile Red with the
holo- complex. A,
fluorescence emission spectra with holo-
complex (5 µM), Nile Red (1 µM), and L-serine (0-50 mM). B, plot of the
change in reciprocal fluorescence emission at 620 nm versus reciprocal concentration of L-serine.
Tryptic cleavage of the subunit in
flexible loop-6 at Arg-188 (51) alters the allosteric
properties of the tryptophan synthase
complex(30, 53) . Fig. 9, A and B, show the fluorescence emission spectra of Nile Red with the
nicked
subunit and ``nicked''
holo-
complex, respectively. The
emission of Nile Red has a larger nonpolar component with the nicked
subunit (Fig. 9A) than with the uncleaved
subunit (Fig. 1A and Fig. 7A). In
contrast, the fluorescence emission of Nile Red with the nicked
complex (Fig. 9B and Table 1) is closely similar to that of the
complex (Fig. 3C and Fig. 7C and Table 3). Spectrum 1 for the nicked
complex in Fig. 9B is clearly not the sum of spectrum 1 for the
subunit in Fig. 1B and spectrum 1 for the nicked
subunit in Fig. 9A, but instead is very similar to the emission
spectra of the holo-
complex and the
holo-
subunit. This result implies that the site that interacts
with Nile Red in the nicked
subunit is not available in the
nicked
complex.
Figure 9:
Effects of ligands on the emission spectra
of the nicked subunit (A) and of the nicked
complex (B). Conditions are
the same as in Fig. 1. Nicked
subunit or nicked
complex alone (curve 1), in
the presence of 80 mMDL-
-glycerol 3-phosphate (curve 2), in the presence of 50 mML-serine (curve 3), or in the presence of both ligands (curve
4).
-Glycerol
3-phosphate has negligible effects on the Nile Red fluorescence with
either
subunit (Fig. 7A) or nicked
subunit (Fig. 9A).
-Glycerol 3-phosphate has a much
smaller effect on the Nile Red fluorescence with the nicked
holo-
complex (Fig. 9B and Table 3) than with the holo-
complex (Fig. 7C and Table 3). Addition of L-serine greatly alters the Nile Red fluorescence of the
holo-
complex (Fig. 7C) and the nicked
holo-
complexes and (Fig. 9B). Thus Nile Red fluorescence with the nicked
holo-
complex appears to have a
normal response to a
subunit ligand but is desensitized to the
effects of an allosteric
subunit ligand.
We will first discuss evidence for the location of Nile Red
in the tryptophan synthase tunnel in the site and then discuss
how this information can be used to understand the effects of active
site (
subunit) and allosteric (
subunit) ligands and of
cleavage of the
subunit loop-6 on the geometry of the tunnel.
To determine the properties and location of the Nile Red binding site, a number of experimental approaches were used. Titration of tryptophan synthase with Nile Red to determine binding strength and stoichiometry is not possible in the usual manner, because Nile Red is much less soluble in aqueous solution than in nonpolar solvents. This is reflected in a partition coefficient of about 200 between nonpolar organic solvents and water(17) . By contrast, other fluorophores that have polarity-sensitive emission, such as 1-anilinonaphthalene-8-sulfonic acid, are quite water-soluble due to an ionized group, but only partition significantly into nonpolar solvents in the presence of a counterion, such as an amine(54) . Nile Red may have advantages over other probes that contain charged groups for probing accessible, but nonpolar sites in proteins. The spectral properties of Nile Red are advantageous for the study of pyridoxal phosphate-containing enzymes such as tryptophan synthase. The absorbance and emission of Nile Red, unlike those of most fluorescent probes, occur in a wavelength range that is substantially removed from the broad absorbance and emission peaks of the enzyme. The drawback imposed by the nonpolar nature of the probe is that the range of aqueous concentrations available for an assay is limited.
The location of the bound Nile Red within the
subunit was investigated using forms of the
complex altered specifically by
chemical modification and site-directed mutagenesis. The nonpolar
component of Nile Red emission is significantly reduced by the presence
of a bulky group at position 170 of the
subunit (see C170-NEM,
C170F, C170W on Fig. 2A and Table 1). Replacing
the bulky side chain of Phe-280, which enters the tunnel in this
region, greatly increases the nonpolar component of Nile Red emission
(see F280C and F280S in Fig. 2B and Table 1).
These results provide additional strong support for a location for Nile
Red in this region of the tunnel near Cys-170 and Phe-280.
The
iodide quenching results ( Fig. 4and Table 2) support a
location for the Nile Red binding site in the holo- subunit, since
the Stern-Volmer quenching constant (K
) obtained
for the holo-
complex is quite
similar to that obtained for the holo-
subunit and quite different
from that obtained for the
subunit. The site of interaction of
Nile Red with the
complex is clearly
different from the
subunit site, because the nonpolar component
of Nile Red emission with the free
subunit has a lower emission
maximum (
619 nm, Table 1) and is completely shielded from
iodide quenching (Table 2), indicating a nonpolar site buried in
the
subunit. The possibility that this site is the active site of
the
subunit is consistent with earlier studies which demonstrated
that acrylamide does not quench the fluorescence of indole-3-propanol
phosphate bound to the
subunit(40) . This site appears to
be unavailable to Nile Red in the native or nicked
holo-
complex.
The quenching
observed with the holo- complex
appears largely due to a static rather than a collisional mechanism.
While this precluded use of the quenching data to determine
accessibility of the bound Nile Red quantitatively,
the
results do indicate that the Nile Red is removed from free contact with
soluble KI since KI can collisionally quench Nile Red emission if it
can contact Nile Red. A site removed from open aqueous solvent is
consistent with the
625-nm emission maximum found for this
component (Table 1), which indicates a nonpolar environment.
Furthermore, alteration of the side chain of
subunit residue 280
has similar effects on KI quenching of Nile Red and pyridoxal
phosphate, suggesting that the two fluorophores are close to each other
and to residue 280. The combined results provide evidence that Nile Red
binds in the holo-
subunit in a nonpolar site, such as the milieu
of the tunnel that leads from the active site of the
subunit to
the active site of the
subunit(15) .
Location of Nile
Red in this portion of the tunnel should allow for nonradiative energy
transfer from pyridoxal phosphate to Nile Red, since the spectral
overlap is satisfactory. Analysis of energy transfer efficiency
(illustrated in Fig. 5) yielded an estimated separation distance
range of 14-27 Å for all of the holoenzyme and reduced
forms of the wild type, F280C and F280S complexes, assuming no major orientational effects. This result
is clearly consistent with a Nile Red binding site near the Cys-170
residue in this portion of the tunnel.
Molecular modeling of Nile
Red binding located a possible binding site of adequate volume in the
subunit near pyridoxal phosphate in the open tunnel model (see
``Results'' and Fig. 6). This site is nonpolar and
removed from contact with bulk solvent and thus consistent with the
spectral properties of the Nile Red emission ( Fig. 1and Table 1), the iodide quenching data ( Fig. 4and Table 2), and the reduction in fluorescence observed with active
site ligands (Fig. 7C and 8 and Table 3). The
orientation of Nile Red in this site is adjacent to Cys-170 and is
consistent with the results of cysteine modification and mutational
replacement of Cys-170 shown in Fig. 2A. Because this
site is blocked by intrusion of Phe-280 into the tunnel (see Fig. SI), this site should be more available with a smaller side
chain on residue 280, which is consistent with the increased
fluorescence emission of Nile Red observed upon replacement of Phe-280
with smaller residues (Fig. 2B). In addition this site
dictates a pyridoxal phosphate-Nile Red separation distance that is
consistent with the energy transfer results. Thus, we suggest that the
location of Nile Red in Fig. 6is the most likely binding site
for Nile Red in the
complex. The
holo-
complex in solution probably
contains an equilibrium distribution of open tunnel and closed tunnel
forms. We suggest that Nile Red binds preferentially to the open tunnel
form. Molecular modeling located a second, more restricted site closer
to pyridoxal phosphate in the closed tunnel structure and a site in the
subunit, essentially at the beginning of the tunnel (not shown).
The site located in the
subunit may be the origin of the nonpolar
component of Nile Red interaction with the free
subunit, but
appears to be unavailable in the
complex.
We have previously reported
that cleavage of the subunit at Arg-188 in a flexible loop-6
prevents the transmission of ligand-induced conformational changes from
the
subunit to the
subunit(30, 53) . Our
new finding that this tryptic cleavage desensitizes the
holo-
complex to the allosteric
effects of
-glycerol 3-phosphate on the nonpolar component of Nile
Red emission (Fig. 9B and Table 3) provides
direct evidence that cleavage of loop-6 prevents ligand-induced
communication between the
subunit and a specific site in the
subunit via the tunnel. It is noteworthy that the nonpolar
component of Nile Red emission is enhanced in the nicked
subunit
relative to that in the uncleaved
subunit (Fig. 9A
versus 7A) but is not altered in the nicked
complex (Fig. 9BversusFig. 7C). These results indicate
that proteolytic cleavage of the separate
subunit alters the
conformation and makes the nonpolar interior more accessible to Nile
Red, whereas the same cleavage of the complex does not alter the
accessibility of Nile Red in the nicked
complex. These results imply either that the conformation of the
nicked
subunit is stabilized by interaction with the
subunit or that the nonpolar interior of the nicked
subunit is
less accessible to Nile Red in the nicked
complex or both.
Other site ligands (D- and L-tryptophan,
indole, benzimidazole, and L-serine + benzimidazole) also
markedly decrease the fluorescence intensity of Nile Red with the
holo-
complex (Table 3).
Because these ligands bind at or near the putative Nile Red binding
site shown in Fig. 6, these
site ligands might be expected
to be competitive inhibitors of Nile Red binding. However, fluorescence
titration results indicate that indole and L-tryptophan are
noncompetitive inhibitors of Nile Red (Fig. 3C). A
probable explanation for these results is that Nile Red binds
reversibly to the open tunnel form of the
site and is released
upon addition of
site ligands that induce the closed tunnel
conformation of the
subunit that does not bind Nile Red. Thus
Nile Red and
site ligands do not compete because they do not bind
to the same form of the enzyme. Similarly, the substrates used in the
assay of activity in the
reaction (indole-3-glycerol
phosphate and L-serine) induce the closed tunnel conformation
of the
subunit that does not bind Nile Red. Thus Nile Red cannot
bind and cannot inhibit the enzyme under steady-state reaction
conditions. Rapid kinetic experiments under single turnover conditions
using enzyme preincubated with Nile Red might detect effects of Nile
Red in the tunnel on the initial rate.