(Received for publication, September 7, 1995; and in revised form, January 29, 1996)
From the
A reversible thermally induced conformational transition of the
subunit of tryptophan synthase from Salmonella
typhimurium has been detected by use of the pyridoxal 5`-phosphate
coenzyme as a spectroscopic probe. Increasing the temperature converts
the major form of pyridoxal 5`-phosphate bound to the
subunit from a ketoenamine species with
at
410 nm to a enolimine species with
at 336 nm (T
=
43 °C) and results in loss of
the circular dichroism signal at 410 nm and of fluorescence emission at
510 nm. The results indicate that increasing the temperature favors a
conformer of the enzyme that binds pyridoxal 5`-phosphate in a more
nonpolar environment and leads to loss of asymmetric pyridoxal
5`-phosphate binding. The internal aldimine between pyridoxal
5`-phosphate and the
-amino group of lysine 87 is not disrupted by
increased temperature because sodium borohydride treatment of the
enzyme at either 15 or 60 °C results in covalent attachment of
[4`-
H]pyridoxal 5`-phosphate. The thermal
transition of the
subunit below 60 °C produces
reversible thermal inactivation (T
=
52 °C) and occurs at a much lower temperature than the major
reversible unfolding at
80 °C (Remeta, D. P., Miles, E. W.,
and Ginsburg, A.(1995) Pure Appl. Chem. 67, 1859-1866).
Our new results indicate that the 410 nm absorbing species of pyridoxal
5`-phosphate is the catalytically active form of the cofactor in the
subunit and that the low temperature reversible
conformational transition disturbs the active site and causes loss of
catalytic activity.
Pyridoxal 5`-phosphate (PLP) ()serves as the coenzyme
for many enzymes that catalyze a wide variety of reactions involved in
the metabolism of amino acids (racemization, transamination,
-elimination,
-replacement, decarboxylation,
etc.)(1, 2) . In every PLP-dependent enzyme, the PLP
coenzyme forms an internal aldimine with the
-amino group of a
lysine residue (see structures in Fig. 1A under
``Results''). This internal aldimine usually exhibits an
absorption maximum at 410-430 nm attributed to the resonance
stabilized ketoenamine form II in Fig. 1A(3, 4, 5) . The
dipolar structure II is favored by a more polar environment. Most PLP
enzymes also have peaks at 330-340 nm, which may represent the
enolimine tautomer (I in Fig. 1A), a neutral
species that prefers a nonpolar
environment(3, 4, 5, 6) . Reactions
with substrates or inhibitors or changes in pH often lead to marked
alterations in the spectrum of the enzyme-bound
PLP(2, 5) . Thus PLP serves as a useful chromophoric
reporter group for changes in the PLP binding site in enzymes and for
reactions with substrates and inhibitors.
Figure 1:
Structures and
absorption properties of internal aldimines formed between PLP and the
-amino group of an enzyme lysine residue. A, structures
of the enolimine form I and of the resonance stabilized ketoenamine
form II (see Introduction). B, effect of temperature on the
absorption spectra of the
subunit. Absorption spectra
of the
subunit (1.17 mg/ml in Buffer P) were recorded
after equilibration for at least 10 min at each temperature shown (17,
30, 40, 50, 60, and 70 °C) and at additional temperatures not shown
(21, 25, 35, 45, 55, and 65 °C) using Method
1.
The subunit of tryptophan synthase (EC 4.2.1.20) catalyzes a number
of PLP-dependent reactions including a
-elimination reaction with L-serine () and a
-replacement reaction with L-serine and indole ().
(For reviews see (7, 8, 9) .) PLP
serves as a useful spectrophotometric indicator of alterations in the
PLP binding site of the subunit. For example, the
UV-visible spectra (10, 11) and circular dichroism
spectra (12, 13) of bound PLP are altered by
interaction of the
subunit with the
subunit of
tryptophan synthase to form an
complex and by certain
subunit mutations in the
complex(11) .
The
three-dimensional structure of the tryptophan synthase
complex from Salmonella
typhimurium revealed that the chains are arranged in a nearly
linear
order(14) . The larger
chain
(43,000 M
) in the complex has two domains of about equal
size, designated the N- and C-domains, which are folded in similar
helix/sheet/helix structures. The active sites of neighboring
and
subunits are connected by a buried,
30 Å long
hydrophobic tunnel that passes between the N- and C-domains of each
chain. This unique tunnel serves as a likely conduit of indole
from the
site to the
site. The binding site for the PLP
coenzyme is located between the N- and C-domains at one end of the
tunnel. The single tryptophan in the
chain, Trp-177, is buried in
the N-domain.
We report here that the spectral properties of PLP
bound to the subunit of tryptophan synthase from S. typhimurium are altered by temperature. The results provide
evidence for a reversible thermal transition in the
subunit at a much lower temperature (T
=
41-47 °C) than the temperature (
80
°C) at which the
subunit undergoes a major
unfolding transition (15, 16, 17) . (
)Irreversible inactivation occurs at the higher temperature (T
=
77 °C) in the presence of a
higher salt concentration that causes protein
aggregation(18, 19) . Other studies show that the low
temperature transition results in a small, low temperature endotherm in
differential scanning calorimetry, perturbation in the environment of
Trp-177 but not of tyrosine residues, and loss in the ellipticity of
PLP(15, 16, 17) .
Our finding
that this conformational transition results in loss of activity shows
that the conformational transition has biochemical and functional
relevance.
Fig. 2A shows plots of absorbance data versus temperature for the subunit and for the model
Schiff base. The absorbance changes of the
subunit
occurring below 60 °C were analyzed assuming a simple two-state
equilibrium between two forms of the protein, a low temperature form, N, and a high temperature form, I. The equilibrium
constant for this reaction, K, can be calculated from the
temperature dependence of the absorbance of the solution at any
wavelength using :
Figure 2:
Effect of
temperature on spectral properties and activities of the subunit. A, plot of normalized extinction coefficients versus temperature in °C for the absorbance changes at 410
nm (
) using Method 1 (see Fig. 1B), and
additional data were collected (
) using Method 2. The deviation of
the data obtained by the two methods at higher temperatures may result
from the fact that the enzyme used for Method 1 was expressed by a
plasmid encoding the trpB gene (21) , whereas the
enzyme used in Method 2 was prepared by heat precipitation of the
subunit from the
complex(22) . The data were fitted to and as described under ``Results.'' The derived value
of T
= 43.2 ± 0.8 is given. The
changes in the mM extinction at 410 nm (
) for the model
Schiff base of L-serine and PLP (0.05 mM PLP with 50
mML-serine in Buffer P) were determined from spectra
recorded at different temperatures as described for the enzyme in Fig. 1and are plotted in a similar way. The dashed lines show pre- and post-transition base-lines. B, the
ellipticity changes at 410 nm (
) are plotted versus temperature in °C and fitted to and as described under ``Results.'' The derived T
value is given. C, the ratios of the
observed initial rate of the
subunit in
-elimination (
) () and
-replacement
(
) () reactions (Fig. 3A) to the
predicted rates (dashed lines in Fig. 3B) are
plotted versus temperature in °C. D, the
logarithm of the fluorescence emission at 510 nm upon excitation at 420
nm is plotted versus temperature in °C for the
subunit alone (
),
subunit + L-serine (
), and model Schiff base (
).
Intensities are plotted on a semi-logarithmic scale versus temperature in °C in order to show all of the data on the same
chart and to simplify the temperature dependence of the fluorescence of
the native protein. Curves were fitted to the untransformed data as
explained in the text. Intensities were measured on solutions in Buffer
P containing 61 µM
subunit alone, 3.7
µM
subunit in the presence of 0.1 ML-serine, or 0.05 mM PLP in the presence of 50
mML-serine and were normalized to 3.7 µM PLP. Measurements on the
subunit alone and of
the model Schiff base of PLP with L-serine were made on a
single solution of each after equilibration for 10-15 min at each
temperature. Each measurement on the
subunit in the
presence of L-serine was made on a separate solution after
equilibration at the indicated temperature for 10 min. Two sets of data
were collected in the presence of L-serine. One set was
collected in the presence 50 µM PLP; the other was
collected in the absence of added excess PLP. Each data set gave the
same T
. The temperature of the
subunit alone was gradually increased up to 73 °C and then
cooled to 4.5 °C. The finding that the fluorescence emission of the
previously heated
subunit was 86% that of the
unheated enzyme at 4.5 °C demonstrates that the thermal transition
is largely reversible. The derived values of T
are
given and are marked on the curves by small
arrows.
Figure 3:
Effect of temperature on the activities of
the subunit. A, the initial rates of the
subunit in
-elimination (
) () and
-replacement (
) () reactions
are plotted versus temperature in °C. B, the data
from A are plotted as log of activity versus the
reciprocal of the absolute temperature in K. The low temperature data
were fitted to the Arrhenius equation () as described under
``Results,'' and the derived constants were used to predict
rates at higher temperatures (dashed lines). These values
together with the measured rates were used to calculate the values of
(T) for both reactions, which were then fitted to to derive the values of T
given in Fig. 2C. and were combined to
predict the temperature dependence of the measured activities (solid lines in Fig. 2C).
where A(T) is the absorbance of the solution
and A(T) and A
(T) are the absorbancies of the low and
high temperature forms of the protein at temperature T, where T is absolute temperature in K(26) . The temperature
dependence of A
(T) could not be observed,
because it was masked by the more extensive changes occurring above 60
°C ( Fig. 1and Fig. 2A). For a two-state
unfolding mechanism, the dependence of K on temperature T is given by :
where G(T),
H, and
S are the changes in free energy, enthalpy, and entropy
of the reaction and T
is the midpoint and where
G(T) = 0, assuming
C
= 0. A control experiment with the model Schiff base of
PLP with L-serine showed a linear temperature dependence (Fig. 2A). The temperature-dependent decreases in
absorbance at 410 nm and increases at 336 nm were much smaller than
those observed with the enzyme-bound PLP. Absorbance changes at 410 nm
were fitted to and using the PC-MLAB program
(Civilized Software, Bethesda, MD) to derive values of
H and T
using the assumption that the
absorbance of the native protein A
(T)
showed the same linear temperature dependence as that of the model
Schiff base and that the high temperature form of the protein A
(T) showed a parallel linear dependence.
The fitted curve (T
= 43.2 ± 0.5
°C;
H =
180 ± 30 kJ/mol) is shown
in Fig. 2A.
The
temperature dependence of k is given by the
Arrhenius equation ():
where E is the energy of activation of
the reaction and T is absolute temperature in K. Fig. 3B shows Arrhenius plots of the rates of both
reactions. (
)Both plots are linear below 45 °C as
predicted and yield
E
= 29.2
± 0.6 kcal/mol for the
-replacement reaction and
E
= 31 ± 0.6 kcal/mol for the
-elimination reaction. Above 45 °C the measured rates decrease
markedly from those predicted by linear extrapolation of the plots. If
the enzyme is in an equilibrium between an active, low temperature form (N) and an inactive, high temperature form (I), then
the fraction of the protein (
) that is in the active form at a
given temperature is given by :
and the equilibrium constant for the reaction between N and I is given by :
The temperature dependence of (K)T is again
given by , where T is absolute temperature in K.
The low temperature data in Fig. 3A were fitted to the
Arrhenius equation using PC-MLAB, and the derived constants were used
to predict rates at higher temperatures (dashed lines in Fig. 3B). These values together with the measured rates
were used to calculate the values of (T) for both
reactions, shown in Fig. 3B, which were then fitted to to derive the values of T
, shown in Fig. 2C. and were combined to
predict the temperature dependence of the measured activities (Fig. 3B, solid lines) and the rate ratios (Fig. 2C, solid and dashed lines).
Figure 4: Absorption spectra of PLP in dioxane/water mixtures. PLP (10 mM) was diluted 50-fold into dioxane/water mixtures. The final solutions contained 0.2 mM PLP in the indicated percentage of dioxane by volume.
To establish definitively that PLP is bound to the
subunit at 60 °C in the form of an internal
aldimine, we have reconstituted the apo
subunit with
[4`-
H]PLP as described under ``Experimental
Procedures'' and have treated the enzyme with sodium borohydride
at 15 or 60 °C as described(20) . The absorption spectra of
both treated enzymes exhibited peaks at 318 nm, showing that reduction
of PLP had occurred. However, this result could not distinguish whether
free PLP was reduced to pyridoxine 5`-phosphate or whether the internal
aldimine was reduced to 5`-phosphopyridoxyl lysine because these
products have similar absorption spectra. Acid precipitation of the
proteins followed by analysis of the radioactivity in the supernatant
solutions (Table 1) showed that more than 80% of the
[4`-
H]PLP became covalently attached by reduction
of the
subunit at either 15 or 60 °C. Thus the
PLP must be bound as an internal aldimine at 60 °C and must be
reduced by sodium borohydride. The low but significant amount of
radioactivity in the supernatants of both sodium borohydride treated
samples (Table 1) may be due to dissociation of some of the
cofactor before reduction or to binding of a radioactive impurity that
does not form an internal aldimine.
The PLP coenzyme has been used previously as a sensitive
chromophoric probe to investigate the structure and function of many
PLP-dependent enzymes. However, there have been very few studies to our
knowledge of the effects of temperature on the spectroscopic properties
of PLP-dependent enzymes, of PLP itself, or of PLP derivatives. ()Recent studies from Ginsburg's
group(15, 16, 17)
and the
studies reported here show that PLP serves as a useful probe of the
effects of temperature on the structure and function of the tryptophan
synthase
subunit and
complex.
The values of T for
the absorption and circular dichroism changes for the internal aldimine
forms (in the absence of L-serine) shown in Fig. 2(A and B) vary from
40-43
°C, (
)whereas the values of T
for
reversible inactivation (Fig. 2C) and of T
for fluorescence changes for the external
aldimine forms (in the present of L-serine) (Fig. 2D) are significantly higher (
49-53
°C). Although formation of the external aldimine forms in the
presence of L-serine appears to stabilize the enzyme, (
)the spectral changes and inactivation observed in the
presence and the absence of L-serine probably reflect the same
transition. Our results also indicate that the species of the cofactor
that absorbs at 410 nm exhibits optical activity at 410 nm and emits
fluorescence at 510 nm upon excitation at 420 nm is the catalytically
competent species.
The observed low temperature transition does not
result in dissociation of the internal aldimine between PLP and the
-amino group of Lys-87, because treatment with sodium borohydride
results in covalent attachment of PLP to the protein at both 15 and 60
°C (Table 1). Our results indicate that PLP is bound to the
inactive conformer of the
subunit as the enolimine tautomer of
the internal aldimine (I in Fig. 1A), a neutral
species that prefers a nonpolar environment(3, 4) .
Analysis of the spectral properties of
the bound PLP show that both a change in the tautomeric form of the
internal aldimine ( Fig. 1and 2A) and loss of the
ellipticity at 410 nm (Fig. 2B) occur in the low
temperature transition. Thus, the low temperature transition involves
several reversible changes in the structure of the subunit: 1)
perturbation in the environment of Trp-177, 2) change in the
environment of the PLP, and 3) a conformational transition that
contributes to the low temperature endotherm. This transition must
reflect a small change in structure or a conformational change because
it does not break the internal aldimine of PLP (Table 1) or
result in exposure of the buried tyrosine residues in the
subunit.
The low temperature transition is clearly
biologically relevant because it results in loss of catalytic activity.
An additional important conclusion is that the species of PLP that
absorbs at 410 nm, exhibits optical activity at 410 nm, and emits
fluorescence at 510 nm upon excitation at 420 nm is the catalytically
active form of the cofactor in the
subunit.