From the Groningen Biomolecular Sciences and Biotechnology
Institute and the Department of Biochemistry, University of
Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
The transport across the cytoplasmic membrane and
concomitant phosphorylation of mannitol in Escherichia coli
is catalyzed by the mannitol-specific transport protein from the
phosphoenolpyruvate-dependent phosphotransferase system,
enzyme IImtl. Interactions between the cytoplasmic B and
the membrane embedded C domain play an important role in the catalytic
cycle of this enzyme, but the nature of this interaction is largely
unknown. We have studied the thermodynamics of binding of (i) mannitol to enzyme IImtl, (ii) the substrate analog perseitol to
enzyme IImtl, (iii) perseitol to phosphorylated enzyme
IImtl, and (iv) mannitol to enzyme IImtl
treated with trypsin to eliminate the cytoplasmic domains. Analysis of
the heat capacity increment of these reactions showed that approximately 50-60 residues are involved in the binding of mannitol and perseitol, but far less in the phosphorylated state or after removal of the B domain. A model is proposed in which binding of
mannitol leads to the formation of a contact interface between the two
domains, either by folding of unstructured parts or by docking of
preexisting surfaces, thus positioning the incoming mannitol close to
the phosphorylation site on the B domain to facilitate the transfer of
the phosphoryl group.
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INTRODUCTION |
The transport of carbohydrates from the environment into the
bacterial cell is, in many cases, accomplished by a complex of proteins
that together make up the phosphoenolpyruvate-dependent phosphotransferase system
(PTS)1 (1, 2). This system
consists of a number of transport proteins, each one specific for one
carbohydrate, and a chain of cytoplasmic proteins that ultimately
transfer a phosphoryl group derived from PEP to the incoming
carbohydrate. The first protein in this chain is enzyme I, which
accepts the phosphoryl group from PEP and transfers it to HPr, which in
its phosphorylated form is the substrate for the transport proteins.
All transport proteins of the PTS have a similar architecture,
consisting of a part that binds and transports the carbohydrate and is
embedded in the cytoplasmic membrane and two cytoplasmic parts,
responsible for the phosphorylation of the substrate. The parts may or
may not be covalently bound, and all possible combinations do, in fact,
exist. In the case of the mannitol-specific transporter from
Escherichia coli, termed enzyme IImannitol, all
three parts are covalently linked and are, thus, domains of the same
protein. The phosphoryl group is accepted from P-HPr by His-554 in the
C-terminal A domain and transferred to Cys-384 on the B domain.
Mannitol is bound at the periplasmic side and translocated across the
membrane by the N-terminal C domain, after which the carbohydrate is
phosphorylated by the B domain. The protein is believed to function as
a dimer, both in the membrane and solubilized in detergent (3-9).
It is obvious from the above that domain interactions play an important
role in the catalytic cycle of this enzyme. Lolkema et al.
(10), for instance, have shown that phosphorylation of the B domain
causes a 1000-fold increase in the rate of isomerization of the
mannitol-occupied C domain, the principle event in the transport
process. Further evidence that the B and C domains influence each
other's conformation was obtained from an analysis of the binding
kinetics of mannitol to the wild-type enzyme, the subcloned C domain,
and a number of mutants substituted at the position of the second
phosphorylation site (10, 11). However, in the absence of knowledge of
the three-dimensional structure in the intact complex, the nature of
the interaction is still largely unknown. Thermodynamic data can give
additional information, both on the nature of the interaction and also
on the extent of the structural changes that are involved. We have
chosen isothermal titration calorimetry (ITC) in this study to develop
the required thermodynamic data.
In this study, we determine the energetics of mannitol binding to
EIImtl by ITC in inside-out membrane vesicles,
i.e. under conditions that closely resemble the native state
of the protein. To study the effect of phosphorylation, the binding of
a substrate analog, perseitol, which cannot be transported or
phosphorylated (12), to EIImtl in the phosphorylated and
unphosphorylated state is determined and compared with binding of
mannitol. Finally, the effect of removal of the A and B domains on the
functioning of the C domain is investigated. In all of these cases, the
experiments have been performed at various temperatures, enabling us to
calculate
Cpoobs. This
parameter has been shown to correlate with the change in
solvent-exposed groups before and after the binding reaction (see,
e.g., Refs. 13-15) and can, therefore, be used to monitor structural rearrangements taking place in the enzyme upon binding of
the substrate.
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EXPERIMENTAL PROCEDURES |
Materials--
D-[1-14C]Mannitol (2.04 GBq/mmol) was obtained from Amersham Pharmacia Biotech;
D-[1-3H]mannitol (976.8 GBq/mmol) was from
NEN Life Science Products. Perseitol was obtained from Pfanstiehl
Laboratories Inc., Waukagan, IL. TPCK-treated trypsin was from
Worthington or Sigma, and bovine pancreatic trypsin inhibitor was from
Sigma. Enzyme I and HPr were purified as described previously (16, 17).
Decylpoly(ethylene glycol) 300 was synthesized by B. Kwant at the
Department of Chemistry, University of Groningen. All other
chemicals were of the highest purity available.
Growth of Bacteria and Preparation of Membrane
Vesicles--
Membrane vesicles containing large amounts of
EIImtl were obtained from E. coli strain LGS322
(F
thi-1, hisG1, argG6, metB1, tonA2, supE44,
rpsL104, lacY1, galT6, gatR49, gatA50,
(mtlA'p),
mtlDc, D(gutR'MDBA-recA)), which contains a
chromosomal deletion for the wild type mtlA gene,
transformed with the inducible plasmid pMamtlA (18), encoding for
EIImtl. A single colony was used to inoculate 10 ml of LB
medium (10 g/liter Bactotryptone, 5 g/liter Bactoyeast extract, 10 g/liter NaCl) containing 100 µg/ml ampicillin. This culture was grown at 30 °C to an A600 of approximately 0.6 and
then used to inoculate a 1.5-liter culture in the same medium, which
was grown overnight to an A600 of approximately
3. This was used to inoculate a 60-liter fermenter containing the same
medium. The cells were grown to an A600 of 0.7, induced for 2 h at a temperature of 42 °C and harvested. The
final A600 was 0.85, resulting in a total of
86 g of cells (wet weight). The cells were washed three times with
~2 liters of buffer (50 mM NaPi, pH 7.5, 1 mM NaN3) and stored on ice overnight. From this
preparation, inside-out membrane vesicles were obtained as described by
Lolkema and Robillard (8). The final yield was 100 ml of vesicle
solution containing 30 µM EIImtl. The
preparation was stored in 2-ml aliquots at
80 °C until use.
Approximately 30% of the total protein was EIImtl,
analyzed by SDS-polyacrylamide gel electrophoresis. The orientation of
the protein with respect to the membrane was determined from the
mannitol phosphorylation activity before and after treatment with
trypsin (5) and turned out to be approximately 90% inside-out.
Membrane vesicles not containing EIImtl were prepared from
E. coli LGS322 without the plasmid. In this case, 9 liters
of LB medium (without ampicillin) was inoculated 1:50 and grown to an
A600 of 0.9 and then induced in the manner
described above. Membrane vesicles were prepared as described for the
cells containing the plasmid.
PEP-dependent Mannitol Phosphorylation--
The
PEP-dependent phosphorylation of mannitol by
EIImtl was determined as described (19).
Concentration Determination--
The concentration of
EIImtl in the vesicles was determined by the mannitol burst
method, which quantitates the amount of phosphorylation sites present
in the preparation (20). 20 nM enzyme I and 1 µM HPr were added to three aliquots of 160 µl of a
standard buffer solution (25 mM Tris-HCl, pH 8.3, 5 mM DTT, 5 mM MgCl2, 5 mM PEP, 7 mM decylmaltoside). Under these
conditions, the kinetics of conversion of mannitol to mannitol-1-P are
slow and limited by the transfer of the phosphoryl group from P-HPr to
the A domain of EIImtl. A 10 mM
[3H]mannitol solution (20 µl) was added to one aliquot.
In this case, the mannitol phosphorylation reaction was started by
adding 40 µl of the vesicle solution and monitored by applying
20-µl aliquots to a small Dowex column at 20-s intervals as described (19). A vesicle solution (40 µl) was added to each of the other two
aliquots and incubated at 30 °C, one for 10 and the other for 30 min
to allow for complete phosphorylation of EIImtl. The assay
was then started by adding 20 µl of the mannitol solution to each
mixture and monitored in the same way. No differences were observed
between the samples incubated for 10 and 30 min in the absence of
mannitol, indicating that phosphorylation of EIImtl is
completed within 10 min. The change in the concentration of mannitol-1-P in time was extrapolated back to t = 0 to
determine the difference in the mannitol-1-P concentration between
samples incubated with and without PEP. This difference equals the
concentration phosphorylation sites on EIImtl in the
preparation, assuming that the transfer of all the EIImtl
phosphoryl to mannitol is rapid and complete. Since EIImtl
contains two phosphorylation sites per monomer, this number is divided
by two to obtain the concentration EIImtl.
Isothermal Titration Calorimetry--
All titrations were
performed using a MCS isothermal titration calorimeter from Microcal. A
similar instrument has been described elsewhere (21). During
experiments, the vesicle solution was thermostatted at the desired
temperature and stirred at 700 rpm. The injection sequence consisted of
30 injections of 3 µl each from a 100-µl syringe, unless indicated
otherwise. Data were analyzed using Origin software from Microcal.
Samples containing vesicles were dialyzed overnight against 1 liter of
buffer (50 mM KPi or Tris/HCl, pH 7.5, 2 mM EDTA, 1 mM DTT) and, if necessary, diluted
in the dialysis buffer before the experiment. Mannitol and perseitol
solutions were prepared from stock solutions in H2O (30.69 and 13.64 mM, respectively) by dilution in dialysis buffer
to minimize artifacts arising from mixing the two different buffer
solutions during the ITC experiment. Control experiments in which the
mannitol or perseitol solution was injected into the buffer were
routinely run and always showed negligible peaks. All solutions
were degassed by gently stirring under vacuum directly before
measurements.
To study the interaction of perseitol with phosphorylated
EIImtl, the procedure was slightly modified. After dialysis
overnight, 200 ml of the dialysis buffer was taken and brought to 7 mM in MgCl2 and 2 mM in PEP. At the
same time, small volumes of a 80 µM HPr and 100 mM PEP solution were added to 2 or 4 ml of the vesicles
solution to give final concentrations of 5.5 µM and 2 mM, respectively. The vesicle solution was then
reequilibrated with the modified buffer by dialysis for 4 h.
Before calorimetric experiments, a small volume of a 3.3 µM enzyme I solution was added to the vesicles solution
to yield a final enzyme I concentration of 120 nM. The
solution was incubated at room temperature for 10 min before loading
the sample into the calorimeter. Because of the low enthalpy of binding
in this reaction, the injection sequence was changed to 16 injections
of 6 µl each.
Vesicles containing only the C domain in the inside-out orientation
were prepared from EIImtl-containing vesicles by
proteolysis, using 10 µg/ml TPCK-treated trypsin for 45 min at room
temperature. To stop the reaction, soybean trypsin inhibitor was added
to a final concentration of 100 µg/ml, after which the normal
dialysis procedure was followed. The success of the treatment was
evaluated by monitoring mannitol phosphorylation activity and by
SDS-polyacrylamide gel electrophoresis. Intact EIImtl runs
at an apparent molecular mass of 56 kDa and the C domain at 30 kDa.
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RESULTS |
Binding of Mannitol to EIImtl--
Fig.
1A shows a typical example of
the raw data obtained from a titration of EIImtl-containing
inside-out vesicles with mannitol. Upon addition of small aliquots of
the mannitol solution, exothermic signals are observed that, after
saturation of the binding, become small and constant. For comparison, a
titration of a solution of a similar concentration of vesicles prepared
from E. coli LGS322 cells without the plasmid encoding for
EIImtl is also shown. The resulting signals are small and
constant, indicating the lack of specific binding of mannitol in this
case. We can, therefore, conclude that the observed effect is due to the specific binding of mannitol to EIImtl.

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Fig. 1.
Raw titration calorimetry data. A,
33 injections of 3 µl each of a 300 µM mannitol
solution into a solution of 15 µM EIImtl in
inside-out membrane vesicles in 50 mM potassium phosphate,
pH 7.5, 2 mM EDTA, 1 mM DTT at 20 °C
(upper trace). For comparison, 13 injections of the same
mannitol solution into a solution of membrane vesicles without
EIImtl are also shown (lower trace). The peaks
result from mixing of the two solutions. B, integrated heats
of injection for the experiment shown in the top panel. The
solid line represents the best fit of the one-set-of-sites
model to the data. The parameters derived from the fit are:
n = 0.67 ± 0.01, Kd = 115 ± 17 nM, and Hoobs = 63.4 ± 0.9 kJ mol 1.
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The integrated heats for each injection were calculated from the areas
under the peaks and, after correction for the heat of dilution of the
mannitol solution, fitted to a mathematical binding model assuming one
set of sites (Fig. 1B). The model uses three parameters: the
stoichiometry of the reaction n, the association constant
Ka, and the enthalpy change associated with binding
Hoobs (21). The heat resulting from
the first injection often was somewhat smaller than heats generated
from subsequent injections, probably due to imperfect filling or
dilution of the mannitol solution at the tip of the syringe during
equilibration of the calorimeter, and was, therefore, not taken into
account during data analysis. The model fits the data well and yields
an estimate of
63.4 ± 0.9 kJ mol
1 for
Hoobs, 115 ± 17 nM
for the dissociation constant Kd, and 0.67 ± 0.01 for the stoichiometry of the reaction at 20 °C under the
conditions given in the figure legend.
The heat capacity increment of the binding reaction,
Cpoobs, can be obtained
from the temperature dependence of the enthalpy of binding. Fig.
2 shows the results of titrations of
EIImtl with mannitol at various temperatures. The
calculated thermodynamic parameters obtained from these data are listed
in Table I. The variation of
Hoobs as a function of temperature is
linear over the temperature interval that we have investigated (Fig. 2,
inset), resulting in a value of
4.0 ± 0.3 kJ
K
1 mol
1 for
Cpoobs in phosphate
buffer.

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Fig. 2.
Integrated heats of injection for the
titration of EIImtl with mannitol in 50 mM
potassium phosphate, pH 7.5, 2 mM EDTA, 1 mM
DTT at 12.6 °C ( ), 14.9 °C ( ), 20.0 °C (×), and
24.9 °C ( ). The solid lines represent the best
fit of the one-set-of-sites model to the data. Inset, the
variation of Hoobs with temperature.
The solid line is obtained from linear least squares
analysis and has a slope of 4.0 ± 0.3 kJ K 1
mol 1.
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Table I
Mannitol binding by EIImtl
Parameters are derived from a nonlinear least squares fit to the data,
using the one-set-of-sites model. Errors are the standard deviations
obtained from the fits.
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In general, there are two terms that contribute to
Hoobs: a term arising directly from
the binding reaction and a term rising from a change in protonation
state of the protein in the complexed and uncomplexed state.
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(Eq. 1)
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NH+ is the number of protons released by
the buffer and
Hbion is the
ionization enthalpy of the buffer (5.3 kJ mol
1 for
phosphate buffer; Ref. 22). To determine whether protons are released
or taken up by the buffer as a result of the binding process, the
experiment was repeated in 50 mM Hepes and Tris buffers at
pH 7.5 (
Hbion 21 and 47.5 kJ
mol
1, respectively). The binding of mannitol becomes less
exothermic when
Hbion increases
(Table I), indicating that protons are released to the buffer, with a
calculated value for NH+/mol of mannitol of
0.45 ± 0.1 at 20 °C. The
Cpoobs for the
interaction of EIImtl with mannitol in Tris buffer is
3.4 ± 0.4 kJ K
1 mol
1.
The Binding of Perseitol to EIImtl--
Perseitol is a
structural analogue of mannitol (Fig. 3)
that binds to EIImtl, but is not phosphorylated or
transported across the membrane (12, 23). It inhibits the transport
activity of the enzyme competitively, indicating that the binding sites
for mannitol and perseitol are the same. The results of ITC experiments
in which EIImtl is titrated with perseitol at temperatures
between 10.5 and 30 °C are presented in Fig.
4 and Table
II. The binding of perseitol can also be
described by a one-set-of-sites model, using parameters that differ
very little from the parameters that were obtained for the binding of
mannitol to EIImtl. The apparent Kd for
perseitol binding is larger by a factor of 1.5-2 compared with
mannitol, whereas the
H of binding is about 5 kJ
mol
1 less exothermic over the entire temperature
interval.
Cpoobs for
binding of perseitol is
3.9 ± 0.2 kJ K
1
mol
1, very close to the value for mannitol binding under
the same conditions. It, therefore, seems that perseitol is a good
model compound to study the changes in binding affinity upon
phosphorylation of EIImtl, since the protein can not be
dephosphorylated by perseitol.

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Fig. 4.
Integrated heats of injection for the
titration of EIImtl with perseitol in 50 mM
potassium phosphate, pH 7.5, 2 mM EDTA, 1 mM
DTT at 10.5 °C ( ), 15.0 °C ( ), 20.0 °C (×), 25.3 °C
( ), and 30.0 °C ( ). The solid lines represent
the best fit of the one-set-of-sites model to the data.
Inset, the variation of
Hoobs with temperature. The
solid line is obtained from linear least squares analysis
and has a slope of 3.9 ± 0.2 kJ K 1
mol 1.
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Table II
Perseitol binding by EIImtl
Parameters are derived from a nonlinear least squares fit to the data,
using the one-set-of-sites model. Errors are the standard deviations
obtained from the fits. All data were recorded in 50 mM
KPi, pH 7.5, 2 mM EDTA, 1 mM DTT.
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The Binding of Perseitol to Phosphorylated
EIImtl--
The binding of perseitol to the phosphorylated
state of EIImtl proved to be difficult to follow by ITC
because we observed only a small enthalpy change associated with the
reaction, resulting in a low signal to noise ratio. In addition,
measurements at temperatures above 25 °C were hampered by
instability of the base line, possibly due to aggregation during the
experiments. Nonetheless, we were able to determine the binding
parameters for the interaction at two different temperatures, allowing
us to roughly estimate
Cpoobs (Fig.
5 and Table
III). Phosphorylated EIImtl
binds perseitol with an affinity comparable to the unphosphorylated enzyme, but the contributions of
Hoobs and
Soobs to the free energy of binding
differ considerably in both cases. In the case of the
non-phosphorylated enzyme, there is a much larger contribution of the
enthalpy of binding to the stabilization of the complex than when the
enzyme is in its phosphorylated state. This is compensated for,
however, by a larger contribution of the entropy term, an effect that
has been termed enthalpy-entropy compensation and has been observed for
a large number of non-covalent interactions (see "Discussion").
Cpoobs of the binding
reaction of perseitol with phosphorylated EIImtl is less
than 1 kJ K
1 mol
1, both in phosphate and
Tris buffers, i.e. less than 25% of the value for the
non-phosphorylated enzyme.

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Fig. 5.
Integrated heats of injection for the
titration of phosphorylated EIImtl with perseitol in 50 mM potassium phosphate, pH 7.5, 2 mM EDTA, 1 mM DTT at 20.5 °C ( ) and 25.3 °C ( ). The
solid lines represent the best fit of the one-set-of-sites
model to the data.
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Table III
Perseitol binding by phosphorylated EIImtl
Parameters are derived from a nonlinear least squares fit to the data,
using the one-set-of-sites model. Errors are the standard deviations
obtained from the fits. Phosphorylation was achieved in situ
by the presence of 5 µM HPr, 120 nM enzyme I,
and 2 mM PEP as described under "Experimental
Procedures."
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The Binding of Mannitol to Trypsin-treated
EIImtl--
Mild trypsinolysis of
EIImtl-containing membrane vesicles leads to the formation
of two fragments of approximately equal molecular weight (5), an
N-terminal fragment, the C domain of EIImtl, responsible
for mannitol binding and transport across the membrane, and a
C-terminal fragment consisting of the cytoplasmic A and B domains. At
longer exposure times, trypsin fully degrades the A and B domains, but
the C domain is protected from degradation, apparently as a result of
its membrane environment. Removal of the A and B domains leaves the
structural integrity and transport capability of the C domain intact,
since upon complementation with a purified combination of the B and A
domains, IIBAmtl, the activity of the system is restored
(24). We have used this approach here to eliminate the A and B domains
of EIImtl, enabling us to study the influence of domain
interactions on mannitol binding. The results are shown in Fig.
6 and Table
IV. Removal of the A and B domains
results in an increase in Kd to values ranging from
200 to 400 nM, depending on temperature (Table IV).
Hoobs is around 57 kJ
mol
1 and does not vary much with temperature compared
with the wild type enzyme. The resulting value for
Cpoobs is
0.5 ± 0.2 kJ K
1 mol
1, much smaller than in the in
the intact enzyme with the A and B domains present. All values
determined for
Cpoobs in
this study are listed in Table V.

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Fig. 6.
Integrated heats of injection for the
titration of C domain of enzyme IImtl obtained from
trypsinolysis (IICmtl) with mannitol in 50 mM potassium phosphate, pH 7.5, 2 mM EDTA, 1 mM DTT at 9.1 °C ( ), 19.9 °C ( ), and 24.9 °C
(×). For clarity, not all data are shown. The solid
lines represent the best fit of the one-set-of-sites model to the
data. Inset, the variation of
Hoobs with temperature. The
solid line is obtained from linear least squares analysis
and has a slope of 0.5 ± 0.2 kJ K 1
mol 1.
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Table IV
Mannitol binding by IICmtl, prepared from EIImtl by
trypsinolysis
Parameters are derived from a non-linear least squares fit to the data,
using the one-set-of-sites-model. Errors are the standard deviations
obtained from the fits.
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Table V
The observed heat capacity increment,
Cpoobs, and the theoretical number of
residues involved in substrate binding, Rth
Cpoobs was calculated from the
data listed in Tables I-IV; Rth was calculated as
described (15).
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DISCUSSION |
Validity of the One-set-of-sites Model for Binding of Mannitol to
EIImtl--
To be able to quantitatively interpret the
data obtained from isothermal titration calorimetry experiments, one
must choose a mathematical model to analyze the data. Previous binding
studies on substrate binding by EIImtl using equilibrium
dialysis or filtration techniques indicated that there are two binding
sites for mannitol on the EIImtl dimer, a high affinity
site (HA) with a Kd of approximately 45-600
nM and a low affinity site (LA) with Kd
values of 5-10 µM (25-27). Binding studies using flow
dialysis resulted in values ranging from 45 to 212 nM for
the HA binding site (11, 12), whereas the LA site could not be assessed
with this technique. Based on this information, a binding model
describing two possible binding sites with different affinities would
seem to be the appropriate choice. However, attempts to fit the ITC
data with such a model did not give sensible results and were,
therefore, not further pursued. An explanation for this apparent
discrepancy may be found in the values of the parameter c,
the quotient of the concentration of sites in the calorimetric cell and
the Kd,c should be between 1 and 1000 to be able to
calculate the thermodynamic parameters reliably (21). Assuming a value
of 5.2 µM for the Kd of the LA site
(27), the value of the c parameter for this site is just
above 1 in our experiments. For comparison, the values of c
calculated from the data in Table I range from 32 to 210 and,
therefore, it is not surprising that the observed dependence of the
enthalpy on the molar ratio of mannitol and EIImtl is
totally dominated by binding to the HA site. Further support for our
interpretation comes from the fact that the observed dissociation constants for the HA site in phosphate and Tris buffer are in good
agreement with values that were measured previously in inside-out membrane vesicles containing EIImtl, using other
techniques.
The average value of the stoichiometry calculated from the data in
Table I in the absence of detergent is 0.62 ± 0.06, somewhat higher than the expected value of 0.5 for one HA site per
EIImtl dimer. This is probably due to inaccuracies in the
concentration determination of EIImtl, possibly resulting
from oxidation of a small fraction of the protein. The active site
cysteine of the B domain, Cys-384, is the site most prone to oxidation,
a process that results in the loss of phosphorylation activity but not
substrate binding. Since our concentration determination is based on
the former, this would result in an underestimation of the protein
concentration and, therefore, an overestimation of the binding
stoichiometry. It should be noted that introducing an erroneous
concentration of binding sites into the data analysis does not alter
the values obtained for the Kd and
Hoobs of the reaction (28).
Enthalpy-Entropy Compensation--
Although the range of reported
enthalpies in the reactions investigated in this study is almost 80 kJ
mol
1, the value of
Goobs varies only over a very narrow
range (~6 kJ mol
1) for the entire data set. This
phenomenon is termed enthalpy-entropy compensation and can be
illustrated in a plot of
Hoobs
versus
Soobs (Fig.
7). In the case of complete compensation,
the expected slope of such a plot is the experimental temperature (29).
For our data at 25 °C, the temperature at which we have the most
complete data set with the largest range of
Hoobs and
Soobs, we find a slope of 303 ± 8 K, equal within error to the expected value of 298 K. This is
additional evidence that the model we have chosen to represent our data
is correct. It also implies that the temperature dependence of the
enthalpy and entropy functions are dominated by solvent reorganization,
or more specifically, by the removal of water from surfaces that become
buried upon binding of the substrate.

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Fig. 7.
Enthalpy-entropy compensation for the binding
of mannitol (mtl) and perseitol (ptl) to
EIImtl, P2-EIImtl, and C domain of
enzyme IImtl obtained from trypsinolysis
(IICmtl), shown as a plot of
Hoobs ( ) and
Goobs ( )
versus Soobs at 25 °C
(298 K). Data were taken from Tables I-IV. The solid
line is obtained from linear least squares analysis and has a
slope of 303 ± 8 K. The dotted line represents the
average value of Goobs ( 37.8 kJ
mol).
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Dependence of Ligand Binding on Temperature--
The heat capacity
increment of the binding reaction,
Cpoobs, for the reaction
of EIImtl and mannitol is
4.0 ± 0.3 kJ
K
1 mol
1 in phosphate buffer. Qualitatively,
this estimate can be interpreted as a conformational change of the
transporter, resulting in removal of hydrophobic groups from the
surrounding water (30). For a more quantitative analysis, care should
be taken that the determination of
Cpoobs is not hampered by
protonation effects, which can have a large influence on
Hoobs and
Cpoobs of binding (31).
Simulations have shown that the largest deviations of
Cpoobs from the intrinsic
value are expected at the pKa values of the complex
and of the free protein. They are, at constant pH, a smooth function of
the ionization enthalpy of the buffer and change in sign when the
ionization enthalpies of protein and buffer are approximately equal. In
the case of EIImtl, the most likely candidate to undergo a
change in protonation state is a histidine (11), with an approximate
ionization enthalpy of 30 kJ mol
1. We have determined
Cpoobs in phosphate and
Tris buffers with ionization enthalpies that bracket the ionization
enthalpy of histidine, and found that they differ by no more than 18%
of the lowest value between the two. Therefore we conclude that the
values we have determined are close to the intrinsic value for the
Cpoobs of the binding
reaction of mannitol and EIImtl.
The values for
Cpoobs for
the binding of mannitol and perseitol to EIImtl are large
when compared with other binding processes. For example, the
Cpoobs for binding of
serine to the serine receptor of bacterial chemotaxis is approximately
0.7 kJ K
1 mol
1 (32) and for binding of
glucose to yeast hexokinase a value of
0.2 kJ K
1
mol
1 was observed (33). Larger values are observed when
binding is coupled to folding and/or oligomerization; in the case of
the
Cro repressor,
Cpoobs is
6.4 kJ
K
1 mol
1 (34), and for the binding of the
glucocorticoid DNA binding domain to its DNA site,
Cpoobs is
4.2 kJ
K
1 mol
1 (35). Although EIImtl
is known to form dimers and, therefore, it could be argued that dimerization leads to the large value of
Cpoobs determined here,
we do not believe that this is the case. The monomer-dimer equilibrium
has been monitored by a number of techniques (3-9) and, in all of
these cases, monomerization was observed only at concentrations in the
nanomolar range and only in detergent. The concentrations we have
employed in our study are much higher, and we are working with membrane
vesicles with their natural complement of lipids. Thus, it seems fair
to conclude that EIImtl is in its dimeric form in all
experiments in this study. Consequently, the observed thermodynamic
parameters must result from changes in the EIImtl dimer
going from a free state to a substrate-bound state.
Spolar and Record (15) developed a method to determine the number of
residues involved in a binding reaction from the value of
Cpoobs. Application of
their analysis to our data yields estimates of approximately 50-60
residues for the binding of mannitol and 62 for the binding of
perseitol (Table V). This analysis depends on an average value of the
ratio of polar and non-polar surface area that is buried during the
binding process determined from data for globular proteins. Therefore,
in the absence of structural data, it can only give an indication of
the actual number of residues involved, but it illustrates that a major
conformational change takes place upon binding of either mannitol or
perseitol to the EIImtl-dimer when the protein is embedded
in the cytoplasmic membrane.
The Effect of Phosphorylation and Removal of the B Domain on
Cpoobs--
In the case of perseitol binding
to phosphorylated EIImtl,
Cpoobs could not be
determined accurately, but it is clear that the value is considerably
smaller, although probably still rather large for the binding of a
small molecule to a protein. The data do not permit a reliable
calculation of the number of residues influenced by the binding, but we
can qualitatively state that the number is much smaller when perseitol
binds to phosphorylated versus unphosphorylated enzyme. Thus
phosphorylation of the B domain influences the structural changes of
the enzyme upon binding of perseitol to the C domain.
Removal of the A and B domain from EIImtl by trypsinolysis
decreases
Cpoobs to
0.5
kJ K
1 mol
1. The theoretical value of the
number of residue involved in the binding drops accordingly to a value
of 9 residues, indicating that the conformational changes in the
absence of the B and A domains involve considerably fewer residues than
in the intact enzyme. Since it is known that there is no functional
interaction between the A and C domains, we can attribute this
difference solely to the absence of the B domain. Therefore, a very
significant part of the structural changes occurring in the transporter
upon binding of mannitol reside in the B domain, rather than the C domain.
Domain Interactions in EIImtl--
The results
obtained after phosphorylation and trypsinolysis of the enzyme strongly
suggest a conformational change upon binding of the substrate to the C
domain that is propagated to the B domain. Other evidence for
conformational coupling between the B and C domains of
EIImtl has been obtained from mannitol binding and
transport kinetics and their dependence on chemical changes and
mutations at the active site of the B domain (10, 11). Our data support
those conclusions and also suggest a mechanism for the coupling. The very large value of
Cpoobs and the
enthalpy-entropy compensation behavior indicate that a new
complementary surface is formed upon binding, either from folding of
unstructured parts or docking of preexisting surfaces on the protein,
but, in either case, resulting in the removal of solvent-exposed parts
of the protein from the surrounding water. This is most likely to
happen between the B and C domains of the enzyme, because the next step
in the catalytic cycle of the enzyme is phosphorylation of the incoming
mannitol by the B domain, a process requiring precise positioning of
the phosphoryl group donor and acceptor. This would explain the
decrease in
Cpoobs after
removal of the B domain because one half of the complementary surface
is no longer present. The fact that phosphorylation of the protein also
leads to a large decrease in
Cpoobs can be explained
in two different ways; either the surface is already formed upon
phosphorylation so that binding of substrate is no longer required to
generate complementary surfaces, or the newly formed surfaces cannot
complement due to steric repulsion of the C7 moiety of the perseitol
and the phosphoryl group attached to Cys-384 on the B domain. A
possible way to discriminate between the two possibilities is to study
the thermodynamics of mannitol binding in a series of mutants that
mimic the phosphorylated state of the B domain; these studies are in
progress.