Kinetic Analysis of Tentoxin Binding to Chloroplast
F1-ATPase
A MODEL FOR THE OVERACTIVATION PROCESS*
Jérôme
Santolini
§,
Francis
Haraux
¶,
Claude
Sigalat
¶,
Gwénaëlle
Moal
, and
François
André
¶
From the ¶ Protéines Membranaires Transductrices
d'Energie (CNRS-URA 2096), DBCM-CEA Saclay, bâtiment 532, F-91191 Gif-sur-Yvette Cedex, France and
Section de
Bioénergétique, Département de Biologie Cellulaire et
Moléculaire, Commissariat à l'Energie Atomique-Saclay,
F-91191 Gif-sur-Yvette Cedex, France
 |
ABSTRACT |
The mechanism of action of tentoxin on the
soluble part (chloroplast F1 H+-ATPase;
CF1) of chloroplast ATP synthase was analyzed in the light
of new kinetic and equilibrium experiments. Investigations were done
regarding the functional state of the enzyme (activation, bound
nucleotide, catalytic turnover).
Dialysis and binding data, obtained with 14C-tentoxin,
fully confirmed the existence of two tentoxin binding sites of distinct dissociation constants consistent with the observed
Kinhibition and
Koveractivation. This strongly supports a
two-site model of tentoxin action on CF1. Kinetic and
thermodynamic parameters of tentoxin binding to the first site
(Ki = 10 nM;
kon = 4.7 × 104
s
1·M
1) were determined from
time-resolved activity assays. Tentoxin binding to the high affinity
site was found independent on the catalytic state of the enzyme.
The analysis of the kinetics of tentoxin binding on the low affinity
site of the enzyme showed strong evidence for an interaction between
this site and the nucleotide binding sites and revealed a complex
relationship between the catalytic state and the reactivation process.
New catalytic states of CF1 devoid of
-subunit were detected: a transient overstimulated state, and a dead end complex unable to bind a second tentoxin molecule. Our experiments led to a
kinetic model for the reactivation phenomenon for which rate constants
were determined. The implications of this model are discussed in
relation to the previous mechanistic hypotheses on the effect of tentoxin.
 |
INTRODUCTION |
F0F1-ATP synthases are the purveyors of
ATP in chloroplasts, mitochondria and bacteria. They are bound to
energy-transducing membranes and couple the synthesis of ATP (through
photophosphorylation of ADP) to the dissipation of a protonmotive force
(1, 2). The enzyme consists of two discrete parts, F0 and
F1, interconnected by a stalk. F0 is embedded
in the membrane and behaves as a proton channel. The extrinsic part
F1 bears the six nucleotide binding sites; three of them
are catalytic sites for ATP synthesis (for a review, see Ref. 3).
Depending on species, three or more different subunits compose the
F0 moiety: a(1), b(2), and c(9-12) in Escherichia
coli; a(1), b(1), b'(1), and c(9-12) in chloroplasts. In
mitochondria, the F0 moiety bears additional subunits (4). The F1 part consists of five different subunits:
,
,
,
, and
, with
(3),
(3),
(1),
(1),
(1) as
stoichiometry. It is proposed that the ATP synthase acts as a
proton-driven motor (5-7): in chloroplasts and bacteria, subunit c and
subunits
and
(presumably linked to the c-crown) compose the
rotor, while the extrinsic
3
3 crown
linked to the membranous a subunit by the
and b subunits acts as a
stator (8, 9). The rotation relayed by
would sequentially modify
the three-dimensional structure of the three catalytic sites (10) and
induce the ATP synthesis. Evidence for the rotation of
relative to
the
3
3 crown has been presented in the
case of ATP hydrolysis (11-14) and ATP synthesis (15). As an essential
breakthrough, this model strictly correlates the cooperative mechanism
of the enzyme to the rotation of the
-subunit and thus to the proton
gradient dissipation.
Tentoxin (TTX)1 is a natural
cyclic tetrapeptide
(cyclo-(L-methyl-Ala1-L-Leu2-methyl-
ZPhe3-Gly4))
produced by several phytopathogenic fungi of the Alternaria genus (16-17), which induces chlorosis of many sensitive higher plants
(18). Chlorosis seems to be a consequence of the inhibition of
photophosphorylation. TTX indeed specifically inhibits ATP synthesis in
chloroplasts of sensitive species (19) as well as ATP hydrolysis in
isolated CF1 (20). In addition, TTX was shown to bind the
extrinsic part F1 of the F0F1-ATP
synthase (19). An interesting feature of this toxin is its dual effect:
in vitro and at low concentrations (10
8 to
10
7 M), TTX inhibits ATP hydrolysis and
synthesis either in isolated chloroplasts or in isolated
CF1, while at high concentrations (10
5 to
10
4 M) it strongly stimulates ATPase activity
of the isolated enzyme (21) and leads to a partial recovery of the
coupled activity of membrane-bound ATP synthase (22).
The mechanism of TTX action, for the inhibition as well as for the
reactivation, is still unknown. The number of binding sites involved in
the reactivating effect remains controversial. It has been reported
that CF1 binds two molecules of TTX on distinct sites (23)
presumably located on
-subunit, although this point remains obscure
(24-26). Since the affinity of TTX for these two binding sites was
found different, we suggested that they were respectively related to
the inhibitory and stimulatory activities of TTX (23). Also, Mochimaru
and Sakurai (27) have recently suggested the existence of a third very
low affinity binding site, which would account for the reactivation process.
The affinity of TTX for the inhibitory site has been measured in
various forms of the ATP synthase (20, 28), with limited efforts to
relate it to the functional states of the enzyme (27). Rare
investigations have been carried out on the interactions between the
nucleotide binding sites and the TTX binding sites. TTX at inhibiting
concentration does not interfere with the exchange of tightly bound
nucleotide (29), while it seems to promote the ADP release at high
concentrations (30). Nevertheless, the effect of the presence of
nucleotides and, more generally, of the catalytic state of the enzyme
on TTX binding to its low affinity site has never been studied.
In this report, we show strong evidence for the existence of only two
TTX binding sites on chloroplast ATP synthase in the concentration
range of in vitro assays, and we show that they directly
account for the inhibitory and reactivating effects of TTX on the
activity of CF1 and CF1-
. In addition,
thermodynamic and kinetic characteristics of TTX binding on the
inhibitory site were thoroughly determined. No influence of the dynamic
state of the enzyme on this inhibitory binding has been observed. As regards the reactivation process, kinetic experiments revealed the
existence of two new dynamic forms of CF1-
related to
the presence of ATP: a transient overactivated state and a dead end complex unable to bind a second TTX molecule. We show that TTX binding
to its reactivating site is strongly dependent on the presence of
nucleotide on the enzyme. We propose a kinetic model that accounts for
all of our results, of which most of the constants were determined.
 |
EXPERIMENTAL PROCEDURES |
Enzyme Preparation--
The soluble chloroplast ATPase
(CF1) was extracted and purified from spinach
(Spinacia oleracea L.) leaves in the active form devoid of
its inhibitory subunit
, unless specified. The enzyme was stored at
5 °C in 34% ammonium sulfate buffer as described previously (31) at
a protein concentration of 16 mg ml
1.
CF1-
was activated by preincubation at 20 °C in 20 mM Tricine, pH 8, 3 mM DTT for at least 3 h. When CF1 was used, no activation was performed. Protein
concentration was determined by UV absorption spectroscopy assuming for
CF1-
an extinction coefficient of 0.48 cm2
mg
1 at 278 nm (31).
Binding Experiments--
Samples of 500 µl of DTT-activated
CF1-
at various concentrations (10 nM to 10 µM) were dialyzed in SPECTRA/POR tubing (molecular weight
cut-off = 6000-8000) for 24 h at 37 °C against 50 mM Tris-SO4, pH 8, buffer containing various
concentrations of 14C-TTX (ranging from 10 nM
to 10 µM). 14C-TTX was obtained as described
previously (23) with a specific activity of 52 Ci mol
1.
Equilibrium conditions were checked by measuring in small aliquots the
inner and outer radioactivity at four different times during dialysis.
Each counting was performed twice for 4 min using a Beckman LS 3801 Scintillation Counter. 14C cpm ranged from 500 ± 60 to 5 × 105 ± 200. Free TTX concentration was deduced from
the radioactivity measured outside the dialysis tubing, and bound TTX
concentration was calculated from the difference between the
radioactivity measured inside and outside the dialysis tubing. No
significant loss of activity occurred within 24 h in these
experimental conditions. For TTX concentrations higher than 10 µM, 14C-TTX and CF1-
were
first equilibrated for 3 h at 37 °C. Bound and free TTX were
then rapidly separated using a PD 10 Amersham Pharmacia Biotech
chromatography column, and their concentrations were measured as
indicated above. Binding curve was fitted using Microcal ORIGIN 5.0 (Microcal Software).
Kinetics Dialysis--
500-µl samples of 14C-TTX
at various concentrations were dialyzed at 37 °C in a low cut-off
(Mr = 1000) SPECTRA/POR dialysis tubing against
20 ml of assay buffer (50 mM Tris-SO4, pH 8, 40 mM KHCO3, 0.18 mM
MgSO4). Small aliquots were withdrawn from the outer buffer
at different times, and the radioactivity was measured as described
above and converted into TTX concentration. The volume of the dialysis
tube, and thus the exchange surface, was nearly constant in each
experiment. The outer TTX concentration was plotted as a function of
time and fitted to Fick's law using Microcal ORIGIN 5.0. Control
experiments were performed with 500 µM samples of ADP,
diadenosine 5'-hexaphosphate, and Alcyan Blue, titrated by spectrophotometry.
Steady-state Activity Assays--
DTT-activated
CF1-
(10 nM) was incubated at 37 °C in 50 mM Tris-SO4, pH 8, buffer, in the presence of
TTX at various concentrations for 4 h up to 12 h for the
lowest concentrations. 5 min after the addition of 40 mM
KHCO3 and 0.18 mM MgSO4, ATP
hydrolysis was initiated by the addition of ATP (final concentration 1 mM). The reaction mixture was thermostatted at 37 °C,
and aliquots of 10 µl were taken every 3.5 min (up to 15 min) and
injected into a TSK DEAE 2SW 5-µm analytical high pressure liquid
chromatography column for nucleotide determination. The nucleotides
were separated by isocratic elution with 0.1 M
KH2PO4, pH 4.3, 0.25 M NaCl, at a
rate of 1.0 ml min
1. ADP concentration was determined
from the area of the peak detected at 260 nm. The ATPase activities of
CF1-
in the presence of various concentrations of TTX
were deduced from the plots of ADP concentration as a function of time
and normalized to the control activity without TTX (Fig. 4).
Time-resolved ATPase Activity Assays--
For inhibition kinetic
studies, DTT-activated CF1-
(10 nM) was
incubated for 10 min at 37 °C in a stirred reaction buffer in a
spectrophotometer cuvette. The reaction buffer (50 mM
Tris-SO4, pH 8, 40 mM KHCO3, 4 mM MgSO4, 1 mM phosphoenolpyruvate,
0.3 mM NADH, 0.1 mg/ml lactate dehydrogenase, 0.1 mg/ml
pyruvate kinase) allowed us to couple ATP hydrolysis to NADH oxidation.
The time response of this system, checked by ADP addition in the
absence of TTX, was always significantly shorter than a few seconds.
ATP hydrolysis was started by adding MgATP (final concentration 2 mM) and monitored by absorbance decrease at 340 nm.
In a first series of experiments (Fig. 3a), TTX was added at
various inhibitory concentrations (from 5 nM to 5 µM) 3 min after the addition of MgATP. In another series
of experiments (Fig. 3b), MgATP was added after the
incubation of CF1-
for various times with 75 nM TTX. The resulting kinetics were fitted to Equation 4.
For reactivation kinetic studies, DTT-activated CF1-
(10 nM) was incubated either in the presence or in the absence
of inhibitory concentration of TTX (500 nM). ATP hydrolysis
was started by the addition of MgATP (final concentration 2 mM) and continuously monitored at 340 nm. In a first series
of experiments, TTX was added at various reactivating concentrations
(from 5 to 150 µM) to inhibited CF1-
, 3 min after the addition of MgATP (Fig. 6a). Similar
experiments were achieved by adding MgATP after the addition of
reactivating concentrations of TTX (Fig. 6b). The decay
phases of the kinetics of Fig. 6, a and b, were
fitted to a primitive function of Equation 14. The resulting apparent
rate constants kapp were fitted to Equation 13,
which allowed the determination of k+,
k
, and K. Other protocols are
detailed in the text.
 |
RESULTS |
Binding of 14C-Radiolabeled TTX to
CF1-
--
Fig. 1 shows
the amount of 14C-TTX bound to CF1-
, when
equilibrated at 37 °C, as a function of the concentration of free
14C-TTX. The data were fitted to a model based on multiple
independent binding sites. The fit clearly shows the existence of two
binding sites of different affinities (Kd1 = 50 ± 20 nM; Kd2 = 80 ± 20 µM) in the concentration range investigated (between 10 nM and 1.2 mM). The two dissociation constants
differ by about 3 orders of magnitude, and the binding of TTX to
CF1-
can therefore be considered to occur in a
sequential mode.

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Fig. 1.
14C-tentoxin bound to
DTT-activated CF1- as a function of free
14C-tentoxin. Conditions were as described under
"Experimental Procedures." CF1- ranged from 10 nM to 10 µM (depending on TTX concentration).
Nonlinear fitting is shown (solid line; see
Equation 1) using a two-independent site model (Kd1 = 50 ± 20 nM; Kd2 = 80 ± 20 µM).
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Monomeric Behavior of TTX at High Concentrations--
To validate
the present analyses, which imply that TTX is monomeric in the reaction
medium, we examined its rate of diffusion through a dialysis membrane
of appropriate cut-off (see "Experimental Procedures"), at
different concentrations. This diffusion indeed is expected to follow
Fick's law only if molecules do not form concentration-dependent aggregates. Fig.
2 (inset) shows exponential kinetics of diffusion of 14C-TTX, as expected from Fick's
law. The kinetic data, normalized to the equilibrium concentration,
were identical for each initial concentration inside the dialysis tube
between 1.8 and 500 µM. The initial rate of diffusion of
14C-TTX was thus found proportional to its internal
concentration (Fig. 2, main panel). To check the dialysis
membrane cut-off, we controlled the rate of diffusion of several
molecules of different molecular masses, at internal concentrations of
500 µM. Their initial rates of diffusion were determined
and plotted as a function of their concentration in Fig. 2. ADP
(Mr = 427) diffused at the same rate as TTX
(Mr = 414.5). Conversely, diadenosine
5'-hexaphosphate (Mr = 996) diffused at a 4-fold
lower rate and Alcyan Blue (Mr = 1299) did not
diffuse at all. This shows that TTX does behave as a monomer, even at
500 µM. This implies that the reactivation of
CF1-
by high TTX concentrations (Figs. 5 and 6) is not
due to some artifact and should be interpreted as the specific binding of a second TTX molecule on its low affinity site.

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Fig. 2.
Diffusion rate of TTX through dialysis
membrane as a function of TTX concentration. Inset,
diffusion kinetics were determined for initial inner TTX concentrations
between 1.8 ( ) and 500 µM ( ) as described under
"Experimental Procedures." The profiles were fitted with the
equation C/Cequilibrium = (1 e t/ ). A fitted curve
(solid line) is shown with = 580 min ( )
and = 551 min ( ). The main panel shows resulting rate
constants plotted versus TTX concentration. In order to
check the cut-off of the dialysis tubing, the same experiments were
performed with 500 µM ADP ( ; Mr = 427), diadenosine 5'-hexaphosphate (AP6A)
( ; Mr = 996), and Alcyan Blue ( ;
Mr = 1299).
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Kinetics of Inhibition of CF1-
by TTX--
The rate
of inhibition by TTX was investigated by injecting TTX to a solution of
CF1-
during ATP hydrolysis, continuously monitored by
ADP-dependent NADH oxidation. Typical recordings of
absorbance at 340 nm are displayed in the inset of Fig.
3a. The absorbance followed a
single exponential decrease superimposed to a linear decay. Therefore,
the experimental data were satisfactorily fitted to Equation 4. The
apparent rate constant kapp of the exponential
kinetics of inhibition was derived for each TTX concentration. As
expected from a unique binding site model, kapp was
found to depend linearly on TTX concentration (Fig. 3a).
According to Equation 3, the slope of the straight line gives the
forward rate constant kon of the binding of TTX to the inhibitory site. The value of kon at
37 °C was found to be 4.7 × 104
M
1 s
1, i.e. 7 times
higher than the value estimated by Mochimaru and Sakurai (27) by
experiments at room temperature.

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Fig. 3.
Kinetics of TTX binding to the high affinity
site. Conditions were as described under "Experimental
Procedures." a, apparent rate constant
(kapp, ) of ATPase inhibition as a function of
TTX concentration. The forward binding rate constant
(kon = 4.7 × 104 ± 2 × 103 s 1) was directly determined from the
slope of the graph (Equation 5). Inset, typical time courses
of ATP hydrolysis after the addition of TTX at 50 nM
(1), 300 nM (2), and 1500 nM (3). Every kinetic profile was fitted to
Equation 4. b, initial ATPase activity ( ) as a function
of incubation time with TTX. Similar experiments are shown as in
a but with 75 nM TTX added at various times
before ATP. The activities were normalized to the control without TTX
(5 ± 1 µmol of ATP/min/mg). Data were fitted to Equation 2,
which gave kapp = 4 10 3 ± 10 3 s 1 and then kon = 6 × 104 ± 1.5 × 104
M 1 s 1 (Equation 3 neglecting
koff). Inset, typical time courses of
ATP hydrolysis after 1 min (1), 5 min (2), and 10 min (3) of incubation, fitted to Equation 4.
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The rate of binding of TTX on the inhibitory site was also measured in
the absence of ATP. CF1-
was incubated in presence of 75 nM TTX for various times. Then ATP was added, the
corresponding kinetics were recorded (Fig. 3b,
inset), and the initial rate of ATP hydrolysis was
determined. This rate was plotted as a function of the time of
incubation with TTX (Fig. 3b). The kinetics of inhibition so
obtained were fitted with a monoexponential function (Equation 2).
Taking into account the TTX concentration and assuming that the
koff is negligible, the derived
kapp gave rise to a forward rate constant
kon of 6 × 104
M
1 s
1. This value is close to
that determined above in the presence of ATP.
Comparison of the Effects of TTX on the ATPase Activity of
CF1 and CF1-
--
Since the rate constant
kon of TTX for the inhibitory site is not very
high, long times of incubations are required to observe the full effect
of low concentrations of TTX (below 100 nM), even at
37 °C. For this reason, we investigated the inhibitory effect of low
concentrations of TTX under conditions similar to those previously
reported (32), except that the time of incubation with TTX was raised
in order to reach the binding equilibrium (see "Experimental
Procedures"). The results are displayed in Fig.
4a. The data points were
fitted to Equations 6 and 7, giving a Kd for the
inhibitory site of about 8 nM. This Kd is lower than that previously determined (38 nM) for
shorter incubation times (32). The resulting value of the rate constant
of dissociation koff is 3.7 × 10
4 s
1. This value confirms that
koff was negligible with respect to kon[T] above.

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Fig. 4.
ATPase activity of CF1 and
CF1- as a function of TTX concentration. Conditions
were as described under "Experimental Procedures." Activities were
normalized to the control without TTX (5 ± 1 µmol of ATP/min/mg
for DTT-treated CF1- and 250 ± 50 nmol of
ATP/min/mg for CF1). a, experimental points
( ) were determined at equilibrium after the incubation of 5 nM of DTT-activated CF1- with various
concentrations of TTX in the inhibitory range. The right
part of the curve corresponds to the nonlinear fitting
previously determined in the reactivatory range (32). The experimental
data were fitted to Equations 6 and 7. Kd1 = 8 ± 1 nM; residual activity V1 = 5%
of the control. b, experimental points ( ) were obtained
in the same conditions with 50 nM of untreated
CF1. The left part (up to 1 µM of TTX) was fitted to Equations 6 and 7;
Kd1 = 13 ± 1 nM; residual activity
V1 = 3%. The right part
(from 1 µM to 1 mM TTX) was fitted with
Equation 8. See text for details.
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The same protocol was used to investigate the effect of TTX on ATPase
activity of native CF1 for concentrations ranging from 1 nM to 1 mM (Fig. 4b). The affinity
of TTX for the inhibitory site of CF1
(Kd = 13 nM) was found almost identical
to the one determined for CF1-
. The lack of the
-subunit and the DTT activation have no effect on the inhibition
processes of TTX. Since the solubility of TTX is limited to 3.5 mM and the monomeric state of TTX is uncertain above 1 mM, the investigation of its effect on CF1
above 1 mM would be of little interest. Therefore, we only
measured the reactivation of the ATPase for TTX concentrations up to 1 mM. Since no plateau of CF1 activity was
reached, we were not able to precisely determine the dissociation
constant for the second, loose site. Whatever it may be, the
Kd varied from 700 µM to 2.7 mM for a reactivation level set from 5- to 20-fold the
control. As a result, the binding of a second TTX molecule appears
quite different between CF1 and CF1-
.
Kinetics of Reactivation of ATPase Activity of CF1-
by TTX Critically Depend on the ATP Preincubation Time--
We have
investigated the kinetics of reactivation of CF1-
by
TTX. The enzyme was introduced in the spectrophotometric cuvette with
TTX at 500 nM to fill the inhibitory site and in the
presence of an enzymatic system coupled to NADH oxidation. In order to check the effect of nucleotides on the TTX-dependent
reactivation process, the enzyme was incubated with ATP for various
times (from 2 s to 10 min) before the addition of a reactivating
concentration of TTX. As expected, the ATPase was inhibited by 500 nM of TTX (6-7% of the control). Reactivation was then
initiated by adding 20 µM of TTX (Fig.
5). Surprisingly, a biphasic profile was
observed for short preincubation time with ATP. It consisted of a fast rise followed by a slow monoexponential decay. This behavior
progressively disappeared with increasing ATP incubation time to
finally give rise to a simple monophasic reactivation.

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Fig. 5.
Kinetics of ATPase reactivation for different
incubation times with ATP. Conditions were as described under
"Experimental Procedures." CF1- (10 nM)
was inhibited by 500 nM TTX and incubated for various times
(9 s (1), 30 s (2), 60 s
(3), 120 s (4), and 300 s
(5)) with 2 mM MgATP. ATP hydrolysis was
monitored after a 20 µM TTX addition. Instantaneous rates
were obtained from the first derivative of ATP hydrolysis profiles.
They were normalized to the control without TTX (6 ± 1 µmol of
ATP/min/mg).
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Rate of Reactivation of CF1-
by High Concentrations
of TTX Added after ATP Preincubation--
TTX-inhibited
CF1-
was incubated with ATP during 3 min (conditions
leading to a monophasic profile of reactivation; Fig. 5), and high
concentrations of TTX (5-150 µM) were then added. A
progressive recovery of ATPase activity was observed. The kinetics of
recovery of activity could be satisfactorily fitted to a
monoexponential function (Fig.
6a). Surprisingly, the rate
constant kapp was not a linear function of the TTX
concentration. It was even found to decrease with TTX concentration
(Fig. 6c). This behavior is not that expected from
reactivation kinetically controlled by TTX binding on the low affinity
site.

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Fig. 6.
Apparent rate constant of TTX binding to the
loose site of CF1- as a function of TTX
concentration. a, conditions were as described under
"Experimental Procedures." CF1- (10 nM)
was inhibited by 500 nM TTX and preincubated for 3 min with
2 mM MgATP. ATP hydrolysis was monitored after the addition
of TTX (concentrations ranging from 5 to 150 µM).
Instantaneous rates were obtained from the first derivative of ATP
hydrolysis profiles. They were normalized to the control without TTX
(6 ± 1 µmol of ATP/min/mg). Typical time courses are displayed
for the following TTX concentrations: 150 (1), 100 (2), 75 (3), 30 (4), and 15 µM (5). kapp values were
obtained by fitting each kinetic profile to Equation 2. b,
same experiments as in a, but MgATP was added to the enzyme
immediately after TTX (concentrations ranging from 5 to 150 µM). Typical time courses are represented for the
following TTX concentrations: 150 (1), 100 (2),
60 (3), 25 (4), and 10 µM
(5). kapp values were obtained by fitting
each kinetic profile with Equation 2. c, apparent rate
constants obtained from a and b as a function of
TTX concentration. Data obtained with ATP preincubation ( ) were
fitted to Equation 13 (k+ = 2 ± 1 × 10 2 s 1, k = 1 ± 0.15 × 10 2 s 1,
Kd = 10 ± 13 µM). The
kapp value obtained without preincubation ( ) was
found to be almost constant (kapp = 8 ± 1 × 10 3 s 1).
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Rate of Reactivation of CF1-
by High Concentrations
of TTX without ATP Preincubation--
We also checked the kinetics of
reactivation obtained at different TTX concentrations, added together
with ATP (Fig. 6b). The kinetics were this time clearly
biphasic; the activity passed through a transient overactivation and
then decayed to a plateau similar to the one obtained with long ATP
preincubation (Fig. 6a). We did not try to analyze the fast
rising phase, because its kinetic resolution was not good enough.
However, the slow phase has been fitted to a monoexponential function.
Contrary to what has been calculated in the kinetic analysis of Fig.
6a, the rate constant kapp was here
independent of TTX concentration (Fig. 6c). Identical
results were obtained when reactivating TTX was added to inhibited
CF1-
at different times before ATP (data not shown).
In other experiments, the kinetics of ATPase reactivation were
investigated as in Fig. 6 by adding high concentrations of TTX, but
directly to the active, noninhibited enzyme. The kinetics of
reactivation also followed the biphasic mode already observed in Fig.
6b. Contrary to what was observed in the previous
experiments, preincubation with ATP several minutes before TTX addition
did not change the kinetics of activation (data not shown).
From these experiments, two different modes of reactivation can be
discriminated on a kinetic basis. The first one, observed when TTX is
added several minutes after ATP (Fig. 6a), is a monophasic rise of activity toward an equilibrium value. The second one is observed when ATP is added after or with TTX (Fig. 6b). It
consists of a fast rise of activity followed by a slow monoexponential decay, leading to about the same equilibrium level as the first mode.
Another major difference between these two modes of reactivation is the
characteristic evolution of kapp as a function of
TTX concentration.
Rapid Release of TTX from the Second Site in the Presence of
ATP--
We have also observed that the release of TTX from the
reactivatory site was very fast in the presence of ATP.
CF1-
was first incubated with TTX 50 µM at
room temperature, for 10 min (a sufficient time for TTX to bind on the
two sites), in the presence as well as in the absence of ATP. Then the
sample was quickly 100-fold diluted in the reaction medium containing
ATP, which keeps the high affinity site filled, and the ATPase reaction
was immediately monitored by NADH spectrophotometry. Since no
significant activity could be detected (data not shown), we concluded
that in the presence of ATP, TTX is released from the low affinity site
in a time shorter than the detection time (a few seconds).
 |
DISCUSSION |
Two Binding Sites Completely Account for Inhibition and
Reactivation of CF1-
by TTX--
Two binding sites with
different Kd values account for the inhibitory and
reactivatory effects of TTX. The Kd values
determined by 14C-TTX binding and kinetic experiments are
compatible. They differ from those determined by Pinet et
al. (23) through binding studies, probably because the conditions
were critically different (buffer at 37 instead of 4 °C). We do not
confirm or invalidate the existence of a third binding site of very low
affinity recently proposed (27). However, we think that it should be
considered very cautiously for different reasons. This third site was
indeed revealed at TTX concentrations above 2 mM, where its
monomeric character was not proved. Therefore, acquisition and modeling
of few binding data in millimolar range, which led to an extrapolated
Kd of 6.3 mM, seem very questionable.
The authors also attribute the very strong level of reactivation
(almost 2000% of the control, instead of the 200-300% generally
obtained) to the filling of a third site. However, they used a
nonactivated CF1, about 20-fold less active than
DTT-treated CF1-
. We confirmed this relative magnitude
of reactivation of CF1 (Fig. 4b), but at 1 mM TTX the absolute activity of CF1 remains
lower than the CF1-
activity. Thus, this huge
reactivation value may be due to the combination of the regular
reactivation with a TTX-induced overcoming of the latent character of
CF1 (21), and not to the existence of a third TTX-binding
site on CF1.
The Dynamic State of CF1 Does Not Influence TTX Binding
at the First Site--
We have quantitatively shown that TTX binding
on the inhibitory site is identical, whether CF1 is
activated or not. Mochimaru and Sakurai (27) also reported that partial
trypsic digestion activation of CF1 had no effect on the
rate of TTX binding on the low affinity site. Moreover, our results
directly demonstrate that ATP addition and the catalytic turnover do
not change the rate of TTX binding at the high affinity site.
Consequently, there is no direct influence of the dynamic state of
CF1 on the TTX binding on the first site. Conversely, it
was previously shown that TTX binding on the inhibitory site modifies
neither the affinity of isolated CF1 for ADP (25) nor the
exchange of tightly bound nucleotide (29). All of these results may
indicate that TTX does not inhibit CF1 by preventing its
activation, normally achieved by the protonmotive force in the
membrane-bound CF0CF1 and mimicked by various
treatments in isolated CF1.
We also pointed out that the rate of exchange of TTX at its first
binding site remains much lower than the turnover rate of the active
enzyme; therefore, nonsaturating TTX concentrations lead to a mixture
of fully inhibited and active enzymes. This explains why in thylakoids,
partial inhibition by a low concentration of TTX did not change the
Michaelis constant of ADP for ATP synthesis (33).
ATP Interferes in a Complex Way with TTX Binding at Its Second
Site--
By contrast, ATP modifies the properties of the low affinity
TTX binding site. We have developed a minimal model to account for all
of our data. The main feature is the progressive change, during
incubation with ATP, in the pattern of TTX-triggered reactivation (Fig.
5). It shifted from a biphasic kinetics with a transient overactivation
(Fig. 6b) to a monotonous rise of activity (Fig. 6a). The simplest way to qualitatively explain this result
is depicted in Scheme 1, where only
enzymes bearing one or two molecules of TTX are represented.

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Scheme 1.
Three-state model of TTX binding to the low
affinity site. T, tentoxin. ET and
ET' are forms of the enzyme bearing one TTX molecule on the
high affinity site. ET' is a dead end complex unable to bind
a second TTX molecule. ETT is a complex bearing two TTX
molecules, the only one that is active. Binding of ATP allows the
transformation of ET into ET'. a,
general case leading to biexponential kinetics. b,
simplified case assuming quasi-equilibrium for TTX binding on the low
affinity site and leading to monoexponential kinetics. In both cases,
the top curve describes the theoretical time
course of ATPase activity without ATP preincubation, and the
bottom curve describes the theoretical time
course of ATPase activity after ATP preincubation. c,
predicted dependence of the apparent rate constant
kapp of the exponential kinetics (case
b) upon TTX concentration (Equation 13). See
"Discussion" and "Appendix" for details.
|
|
In the presence of ATP, the ET form is slowly converted into
a dead end complex, ET', unable to bind a second TTX
molecule. The forward rate can be kinetically controlled either by ATP
binding or by any further conformational change. TTX exchange at the
low affinity site is much faster than the ATP-induced conversion
(Scheme 1a). Thus, one may consider that this equilibrium is
reached at any time and may restrict the analysis to the slow phase of
the kinetics characterized by an apparent rate constant
kapp (Scheme 1b, Equation 14). Without
ATP, the ET complex can bind a second TTX molecule, leading
to an ETT complex, the only state catalytically active.
When ATP is added to inhibited CF1-
with or after
reactivating concentrations of TTX, all of the enzyme is initially in
equilibrium between the ET and ETT states.
Therefore, a burst of activity is produced followed by a slow decay of
activity until the equilibrium, involving the three forms, is reached.
When inhibited CF1-
is incubated with ATP a few minutes
before the reactivating TTX addition, an equilibrium is reached between
ET and ET'; after the TTX addition, the activity
monotonously rises to the same equilibrium as before without giving
rise to a transient overaccumulation of the ETT state. The
kinetics observed in Fig. 6, a-b, are well described by
this model, which also predicts that the slow apparent rate constant
kapp decreases with TTX concentration (Scheme 1,
bottom). This prediction conforms with the data obtained
with preincubation of ATP, but not for those obtained without ATP
preincubation for which the kapp was found almost
independent on the TTX concentration (Fig. 6c). In order to
account for all these results, this model has been refined into a final
model depicted in Scheme 2. We kept the
dead end complex, which is necessary to explain the
kapp decrease in the case of ATP preincubation. But
to explain the invariance of the kapp in the case of
no ATP-preincubation, an irreversible and slow transformation of the
enzymatic state (bearing either one or two toxins) has been introduced
upstream from the states of Scheme 1, now included in the
right part of Scheme 2. Moreover, to account for
a transient overactivation observed even at saturating concentration of
TTX (not shown), we had to introduce an overstimulated state.

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Scheme 2.
Multistate model of TTX binding to the low
affinity site. Tentoxin binding sites are represented by
notches. The left notch represents the
inhibitory (high affinity) site, and the right
notch shows the reactivatory (low affinity) site. When
filled, they are, respectively, shaded in gray
and black. For the loose site, the notch is an
open triangle when TTX is slowly exchangeable and
an open rectangle when TTX is quickly
exchangeable. T, tentoxin. Left
column, states of CF1- in the absence of ATP;
right column, states of CF1- in
the presence of ATP. State A is the active form of the enzyme
catalyzing ATP hydrolysis; states I1, I2, and D
are inhibited forms of the enzyme bearing one TTX molecule; states O
and S are reactivated states bearing two TTX molecules. Two
unidirectional arrows with
a box, quasiequilibrium; single
unidirectional arrow, irreversible conversion;
two unidirectional arrows
without a box, reversible conversion.
Thermodynamic and kinetic constants indicated have been determined from
the experiments. The catalytic activities normalized to the control
(activity without TTX) are displayed inside the different
complexes. See "Discussion" and "Appendix" for details.
|
|
In the absence of ATP, binding and release of TTX to and from the two
sites are slow processes (TTX remains bound on these two sites during
the rapid elution of CF1-
on a column, data not shown).
These facts were taken into consideration by introducing ATP-free
enzymatic states (Scheme 2, left), well characterized by the
binding studies (Fig. 1). However, since the addition of ATP generates
in a rapid and irreversible way new enzymatic states characterized by
fast TTX exchange rates on the loose site, the only states required to
explain the kinetic experiments are the ATP-loaded states.
Let us consider the effect of TTX addition to inhibited
CF1-
. When ATP is added just before, simultaneously
with, or after stimulatory concentrations of TTX to inhibited
CF1-
, the enzyme is first in rapid equilibrium between
an overstimulated state (state O) and an inhibited state (state I).
Then it experiences an irreversible and slow conversion into two states
also in rapid equilibrium (named S and I2, respectively,
and corresponding to the ETT and ET states
described in Scheme 1). State I2 slowly equilibrates with
the dead end state D (corresponding to ET' in Scheme 1). The
global process gives rise to a slow decay of the activity, as observed
in Fig. 6b, toward an equilibrium among three enzymatic
forms. The kapp apparent for this decay (Fig.
6c) is about 0.5 min
1 (8 × 10
3 s
1). This value gives the forward rate
constant of the O
S and I1
I2
transitions. The rate constants are assumed to be the same for both
conversions, in agreement with the fact that kapp does not depend on TTX concentration. Since the inhibited enzyme is
subjected to the same slow conversion as the overstimulated one, this
step does not seem to be directly correlated to the catalytic turnover.
After a long preincubation of the inhibited enzyme with ATP, the enzyme
is in equilibrium between the inhibited state (state I2)
and the dead end state (state D). The addition of stimulatory TTX
induces, as in Scheme 1, a monotonous rise of the ATPase activity until
the equilibrium between the stimulated, dead end, and inhibited enzymatic states is reached (Fig. 6a). The
kapp of this monoexponential growth decreases with
the TTX concentration (Fig. 6c, Equation 13). The data gave
the Kd of TTX for the loose site and the forward and
backward rate constants k+ and
k
(Scheme 1). Due to the existence of the dead
end complex, the Kd2 measured at equilibrium is only
apparent and is a function of the true Kd,
k+, and k
(Equation 15). From Fig.
6c and Equation 13, the true Kd is about
10 µM. Consequently, the apparent Kd2
should be about 30 µM (Equation 15), which is in
accordance with the previous estimate in equilibrium
(Kd2 = 40 µM; Ref. 32).
When TTX at reactivating concentrations was added directly to the
active complex (without TTX on the tight site), the kinetic profiles
did not depend on the preincubation of the enzyme with ATP (data not
shown). It consisted in all cases in a fast overstimulation followed by
a slow decay. This is easily explained if we assume that the active
enzyme (state A) does not experience a transition like states
I1 and O. After the addition of high concentrations of TTX,
with or after ATP, the enzyme immediately goes, through the inhibited
states, to the overstimulated state (state O). Then it goes slowly to
the stimulated one (state S) and reaches an equilibrium between states
D (dead end), S (stimulated), and C (inhibited).
Structural Considerations--
The presently available data do not
give a clear cut structural explanation of our results. First, a high
resolution structure of CF1 is still lacking. Second, there
is only indirect evidence (24, 26) that
-Asp-832 takes part in the
TTX binding site. However, for illustrating the TTX effects in relation
to the CF1 structure, we can take for granted that the TTX
binding site is located in this region and that the structure of this
well conserved domain is equivalent in CF1 and
MF1, although MF1 is TTX-insensitive. A survey
of the structure of MF1 (10) indicates that this region,
located at an
/
interface in the vicinity of the upper
-barrel
of the
-subunit, corresponds to a pocket whose opening critically
depends on the nucleotide occupancy. The
/
pair comprising the
adenosine triphosphate-loaded
-subunit (chains B and F in Protein
Data Bank structure 1cow) presents the most favorable conformation of
this region for TTX binding. This domain is rich in aromatic residues.
One of them,
-Tyr-2922, located in a homologous
sequence, FYLH, is a good candidate to stack with the
methyl-Phe((Z)
) residue of TTX. We have shown that the
characteristics of TTX binding on the first site do not depend on the
catalytic state of the enzyme. This site is certainly not subjected to
turnover-dependent changes of exposure. Considering a
stepped rotative mechanism of CF1 (34), the probability for TTX to bind the most favored site is indeed the same in resting and
dynamic states of the enzyme.
The asymmetry of the complex, and thus of the location of bound TTX,
could also explain the differential effect of TTX on ATP synthesis and
hydrolysis under single site conditions (29). In these conditions, the
enzyme does not run a complete revolution during the experiment, and
thus the effect of TTX could depend on the direction of rotation. In
the direction of ATP synthesis, TTX could block the first step(s) of
the rotation and would therefore inhibit the unisite catalysis. In the
other direction, TTX could block more distal step(s) of the rotation,
allowing the release of the ADP molecule, without apparent effect on
the unisite hydrolysis. This hypothesis could also account for the
ATP-dependent reactivatory behavior of TTX. In the presence
of ATP, inhibited forms of CF1-
would carry out a
fraction of turn and thus modify the relative position of TTX.
Therefore, once a first molecule of TTX is bound, the conformations of
the
/
sites and of the whole structure of CF1-
would depend on the presence of ATP. This conformational variation
would modify the binding properties of the second TTX molecule and
explain why, in the presence of ATP, CF1-
does not pass
through the overstimulated form observed in the absence of ATP. The
present discussion shows that the microscopic states, here revealed by
kinetic experiments, should be taken into account in the further
investigations of the catalytic mechanism of the ATP synthase.
 |
ACKNOWLEDGEMENTS |
Thanks are due to Véronique Mary for
the extraction of the spinach chloroplast F1-ATPase.
14C-TTX was provided by Drs. Jean-Marie Gomis and
Jean-Pierre Noel (Service des Molécules Marquées,
Commissariat à l'Energie Atomique-Saclay).
 |
FOOTNOTES |
*
This work was supported by Ministère de
l'Enseignement Supérieur et de la Recherche Contract ACC-SV5
(interface Chimie-Physique-Biologie) no. 9505221 and by CNRS grant
(Physique-Chimie du Vivant) no. 97N21/0122.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 33 1 69 08 44 32;
Fax: 33 1 69 08 87 17; E-mail: santo{at}dsvidf.cea.fr.
The abbreviations used are:
TTX, tentoxin or
cyclo-(L-N-methyl-Ala1-L-Leu2-N-Me
ZPhe3-Gly4); CF1, chloroplast F1 H+-ATPase; CF1-
, chloroplast F1 H+-ATPase
devoid of
-subunit; DTT, dithiothreitol; 14C-TTX, 14C-methyl-Phe((Z)
)-tentoxin.
2
In CF1 primary sequence numbering.
 |
APPENDIX |
Kinetics of ATPase Inhibition by TTX
The binding of TTX to the high affinity site is described as
follows,
|
(Eq. 1)
|
E is the active form, and ET is the
inhibited one. When the toxin is in excess with respect to the enzyme,
the rate of ATP hydrolysis as a function of time is as follows,
|
(Eq. 2)
|
with
|
(Eq. 3)
|
where V0 and Veq
are, respectively, the initial and final rates of ATP hydrolysis.
The instantaneous concentration of ATP can be deduced from Equation 2
|
(Eq. 4)
|
where
|
(Eq. 5)
|
A, B, and kapp were
derived by fitting the experimental data (Fig. 3a) to
Equation 4. kon can be derived from
kapp using Equation 3.
Inhibition by the First TTX Molecule at Equilibrium and Kd1
Determination
To fit the curve of activity (V) versus TTX
concentration, we account here for the concentration of enzyme, which
is not negligible, and for a possible activity of the ET
complex. The activity is described as follows,
|
(Eq. 6)
|
where
is the fraction of the noninhibited enzyme,
V0 is the activity of E, and
V1 is the activity of ET. In a
noncompetitive model,
can be expressed as follows,
|
(Eq. 7)
|
where Kd1 is the dissociation constant of the
ET complex and [E]0 and [T]0 are
the total concentrations of enzyme and TTX. For a noncompetitive
inhibition, Kd1 is not substrate
concentration-dependent.
Reactivation by the Second TTX Molecule at Equilibrium and
Kd2 Determination
At high concentrations of TTX, the concentration of TTX-free
enzyme is negligible. Therefore, the activity versus
concentration curve is represented as follows,
|
(Eq. 8)
|
where V1 is the activity of ET
complex, V2 is the activity of ETT
complex, and Kd2 is its dissociation constant. In
the presence of a large excess of toxin, [T] can be taken as [T] = [T]0.
Three-state Kinetics of Reactivation by TTX at High Concentration
(Scheme 1)
The following three states are considered: ET, the
enzyme bearing one TTX molecule; ETT, the enzyme bearing two
TTX molecules; and ET', a dead end complex, reversibly
formed from ET in the presence of ATP and unable to bind a
second TTX molecule. This system is displayed in Scheme 1.
Scheme 1b describes a rapid equilibrium between states
ET and ETT, which is formalized by the
equation,
|
(Eq. 9)
|
where K is the equilibrium dissociation constant.
Scheme 1b also displays a slow equilibrium between states
ET and ET' characterized by the forward and
backward rate constants k+ and
k
. The evolution of the whole system is
described by the following differential equation.
|
(Eq. 10)
|
Equation 10 may be written as follows,
|
(Eq. 11)
|
where [E]tot is the total enzyme
concentration. [ET] + [ETT] is an exponential
function of time, as follows,
|
(Eq. 12)
|
with
|
(Eq. 13)
|
y0 and yeq are the
initial and equilibrium values of the sum of [ET] + [ETT], respectively. ETT is here assumed to be
the only active form. Since [ETT] is a constant fraction
of [ET] + [ETT], it obeys the same
exponential law. The rate of ATP hydrolysis is proportional to
[ETT] at any time. It can then be written as follows.
|
(Eq. 14)
|
with
|
(Eq. 15)
|
Equation 14 shows that the time course of the activity depends on
the initial concentration of ETT, which is itself determined by the time of ATP preincubation.
Two extreme situations can be distinguished in Scheme 1.
Situation 1--
If TTX is added at reactivating concentration a
long time after ATP, all forms are present upon TTX addition. The
activity, proportional to [ETT]0, is initially
low and then increases to reach its equilibrium value.
Situation 2--
Without incubation with ATP, ET and
ETT are the only forms initially present. The activity
starts at its maximal level and decreases to its equilibrium value.
Equation 13, which gives kapp as a function of
[TTX] (Scheme 1c) applies to both situations.
The final multistate model (Scheme 2) will be developed from Scheme
1b, which is a simplified form of Scheme 1a in
which the equilibrium between the ET and ETT
states is instantaneously reached.
Multistate Kinetics of Reactivation at High Concentrations of TTX
(Scheme 2)
In Scheme 2, the enzyme may bear zero, one, or two TTX molecules.
The left three ATP-free states will not be considered here. When ATP is
added, the substates initially present are forms A (active, no TTX
bound), I1 (inhibited, one TTX molecule bound), and O
(overstimulated, two TTX molecules bound). States I1 and O
are in rapid equilibrium.
Situation 1--
The incubation of the inhibited enzyme with ATP
for a long time directly leads to the formation of states
I2 and D (ET and ET' in Scheme 1).
The addition of reactivating TTX leads to a situation quite identical
to that described in Scheme 1, with the same prediction, an activity
initially low that increases to reach its equilibrium value, with a
rate constant, kapp, defined in Equation 13.
Situation 2--
When ATP is added after reactivating TTX or, at
the same time, the enzyme is initially under states I1 and
O (state A can be neglected), the system then evolves toward the
formation of states S, I2, and D. The quasi-equilibrium
between states I1 and O is described by the dissociation
constant K0 as follows.
|
(Eq. 16)
|
If [X] = [I1] + [O] and if the irreversible
conversion of states I1 and O, respectively, into states
I2 and S occurs with the same rate constant
ka, the evolution of [X] is given by
the equation,
|
(Eq. 17)
|
where
|
(Eq. 18)
|
Likewise, if [Y] = [I2] + [S], where the
following is true,
|
(Eq. 19)
|
the evolution of [Y] is given as follows.
|
(Eq. 20)
|
Using Equation 19, Equation 20 becomes the following,
|
(Eq. 21)
|
where K, k+, and
k
have the same definitions as in Scheme 1.
Since the following is true,
|
(Eq. 22)
|
Equation 21 becomes the following.
|
(Eq. 23)
|
The initial conditions are [X]0 = [E]tot and [Y]0 = 0. Thus,
Equation 23 becomes the following.
|
(Eq. 24)
|
The solution of this equation is as follows,
|
(Eq. 25)
|
with
|
(Eq. 26)
|
and
|
(Eq. 27)
|
|
(Eq. 28)
|
|
(Eq. 29)
|
and
|
(Eq. 30)
|
If VOver is the activity of state O and
VStim is the activity of state S, the global
ATPase activity V as a function of time is
Vt = VOver[O] + VStim[S], which gives the equation,
|
(Eq. 31)
|
or
|
(Eq. 32)
|
The model is described by the sum of two exponential decays
characterized by two distinct rate constants:
k1, which is [TTX]-dependent (Equation 24), and k2, which is not (Equation 25).
In Situation 2, we took into account the kinetic data collected after 1 min in order to focus on the slow component of the decay (Fig.
6b). The rate constant that characterized this decay proved
to be [TTX]-independent (Fig. 6c) and was thus identified as ka.
 |
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