(Received for publication, August 1, 1995; and in revised form, October 4, 1995)
From the
The and
complexes of F
-ATPase
from a thermophilic Bacillus PS3 were compared in terms of
interaction with trinitrophenyl analogs of ATP and ADP (TNP-ATP and
TNP-ADP) that differed from ATP and ADP and did not destabilize the
complex. The results of equilibrium
dialysis show that the
complex
has a high affinity nucleotide binding site and several low affinity
sites, whereas the
complex has only
low affinity sites. This is also supported from analysis of spectral
change induced by TNP-ADP, which in addition indicates that this high
affinity site is located on the
subunit. Single-site hydrolysis
of substoichiometric amounts of TNP-ATP by the
complex is accelerated by the
chase addition of excess ATP, whereas that by the
complex is not. We further examined
the complexes containing mutant
subunits (Y341L, Y341A, and
Y341C). Surprisingly, in spite of very weak affinity of the isolated
mutant
subunits to nucleotides (Odaka, M., Kaibara, C., Amano,
T., Matsui, T., Muneyuki, E., Ogasawara, K., Yutani, K., and Yoshida,
M.(1994) J. Biochem. (Tokyo) 115, 789-796), a
high affinity TNP-ADP binding site is generated on the
subunit in
the mutant
complexes where
single-site TNP-ATP hydrolysis can occur. ATP concentrations required
for the chase acceleration of the mutant complexes are higher than that
of the wild-type complex. The mutant
complexes, on the contrary, catalyze single-site hydrolysis of TNP-ATP
rather slowly, and there is no chase acceleration. Thus, the
subunit is responsible for the generation of a high affinity nucleotide
binding site on the
subunit in F
-ATPase where
cooperative catalysis can proceed.
F is a hydrophilic portion of H
-ATP
synthase and can catalyze hydrolysis of ATP. Isolated
F
-ATPase is comprised of five different subunits in a
stoichiometry
. Each
of isolated
and
subunits can bind one nucleotide, but
neither of them has ATPase activity (Yoshida et al., 1977a;
Dunn and Futai, 1980; Ohta et al., 1980; Issartel and Vignais,
1984; Hisabori et al., 1986; Rao et al., 1988;
Bar-Zvi et al., 1992). The crystal structure of bovine
mitochondrial F
-ATPase (MF
) (
)(Abrahams et al., 1994) revealed that the
and
subunits have a similar fold alternating in a hexagonal
arrangement around a central cavity containing the
subunit as
expected from previous electron microscopic studies (Gogol et
al., 1989; Fujiyama et al., 1990), and catalytic
nucleotide binding sites reside mostly on
subunits whereas
noncatalytic nucleotide binding sites are mostly on
subunits. The
subunit has at least three stretches of
helices, and two of
them form a coiled coil structure that penetrates the central cavity of
the
structure.
Penefsky and his
colleagues showed that bovine MF has a single high affinity
ATP binding site (Grubmeyer et al., 1982; Cross et
al., 1982). ATP added at substoichiometric amount binds rapidly to
this site and is hydrolyzed slowly (single-site or
``uni-site'' catalysis). This slow hydrolysis is greatly
accelerated by the addition of excess ATP (chase acceleration).
However, in the case of F
-ATPase from a thermophilic Bacillus PS3 (TF
), the single-site hydrolysis of
ATP is much faster than that of MF
, and only a very poor
chase acceleration is observed (Yohda and Yoshida, 1987). Nonetheless,
steady state ATP hydrolysis by TF
does not obey simple
Michaelis-Menten type kinetics but shows apparent cooperativity (Wong et al., 1984; Yokoyama et al., 1989). The attenuated
incorporation of water oxygen into the product P
during
hydrolysis of increasing concentrations of ATP by TF
also
provided a support for cooperative nature of the catalysis (Kasho et al., 1989). We found that TF
hydrolyzed a
substoichiometric amount of trinitrophenyl adenosine triphosphate
(TNP-ATP) slowly, and this slow hydrolysis was accelerated by the chase
addition of excess ATP (Hisabori et al., 1992). These
observations have lead us to the conclusion that TF
, as
well as F
from other sources, has a functional asymmetry
among the three catalytic sites, although TF
molecules,
different from F
from other sources, do not contain any
endogenously bound nucleotide. Moreover, from the analysis of the
nucleotide binding of the TF
ADP 1:1 complex, we
suggested the functional asymmetry among the three catalytic sites
(Hisabori et al., 1994).
TF is unique in its
ability to reconstitute from each isolated subunit. Complexes with
various combinations of subunits, such as
and
(Yoshida et al.,
1977a; Yokoyama et al., 1989) and
(Miwa and Yoshida, 1989; Kagawa et al., 1989) have been
characterized. Most recently, the minimum catalytic unit, whose most
probable subunit composition was
,
was reconstituted on the solid support (Saika and Yoshida, 1995). As
expected, this
complex shows simple
Michaelis-Menten type kinetics when it hydrolyzes ATP. Therefore a
critical question has arisen: is the kinetics of the
complex cooperative or not? Because
the structure of this complex should be a perfect 3-fold symmetry, all
three catalytic sites, as well as three noncatalytic sites, should be
equivalent. If catalysis by this complex obeys noncooperative kinetics,
then one can conclude that kinetic cooperativity observed for
F
-ATPase might be due to structural asymmetry caused by the
presence of single-copy subunits. If it is cooperative, this means that
structural asymmetry introduced by binding of the first substrate is
responsible for the kinetic complication of F
-ATPase.
However, answering the above question by experiment has turned out to
be not as easy as it seemed at first. We had previously reported that
the
complex exhibited cooperative
kinetics (Miwa and Yoshida, 1989), but this observation could be
interpreted in a different way because Harada et al. found
that the complex tended to dissociate into
complexes in the presence of ATP (>6 µM) or ADP
(>30 µM) (Harada et al., 1991). Here, we
report that TNP-ATP (or TNP-ADP) does not destabilize the
complex, and this provides an
opportunity to examine the kinetic properties of the
complex. Comparison of the
characteristics of the
complex
and those of the
complex indicates
that the structural asymmetry of F
-ATPase introduced by the
subunit to the
structure is
responsible for generation of a high affinity nucleotide binding site
where single-site hydrolysis can occur. In addition, the complexes
containing mutant
subunits, whose Tyr-341 was replaced by Leu,
Ala, or Cys, were also compared. Although the affinity of isolated
mutant
subunits to nucleotide are greatly diminished (Odaka et al., 1994), the binding of substoichiometric TNP-ATP (or
TNP-ADP) to the mutant
subunits returned to normal when they were
assembled into the
structure.
Figure 1:
Stability of
the complex in the presence of
TNP-ATP (or TNP-ADP). A, the
complex was preincubated for 1 min in the solution containing the
indicated concentrations of TNP-ATP and subjected to gel filtration
HPLC. The column was equilibrated and eluted with the same solution
used for preincubation. The elution was monitored by measuring the
absorbance at 280 nm. B, the same as A except that
TNP-ADP was used instead of TNP-ATP. C, the same as A except that the
complex was
preincubated with 10 µM TNP-ATP and applied to a gel
filtration column equilibrated with the solution containing the
indicated concentrations of ATP. All nucleotide solutions contained
MgSO
at concentrations equal to those of
nucleotides.
Figure 2:
Binding of TNP-ADP to
(
) and
(
) complexes measured by
equilibrium dialysis. Details of the experiments are described under
``Experimental Procedures.''
Figure 3:
Transition of difference absorption
spectra of the complex induced
by each of the step-wise additions of TNP-ADP. Into 1 ml of 2
µM
complex
solution in 20 mM Tricine-NaOH (pH 8.0) and 2 mM of
MgCl
, 5 µl of 100 µM of TNP-ADP were added
repeatedly in a step-wise manner, and the difference spectra induced by
each addition were recorded. The molar ratios of TNP-ADP to the complex
before and after each addition and the scale of induced difference
spectra are shown in the figure. The difference spectra observed for
the isolated
and
subunits are shown at the top and bottom, respectively, as references. The magnitudes of these
two spectra were changed arbitrarily. Details of the experiments are
described under ``Experimental
Procedures.''
Figure 4:
Single-site hydrolysis of TNP-ATP and its
acceleration by chase addition of ATP by the
and
complexes. Wild-type and indicated
mutant complexes were examined. Single-site hydrolysis of TNP-ATP was
terminated by the addition of perchloric acid (closed circles)
or was chased by addition of 1 µM (open circle)
or 3.3 mM (open squares) ATP. The reaction was
terminated 5 s after chase addition of ATP by the addition of
perchloric acid. Indicated times are those when perchloric acid was
added. Details of the experiments are described under
``Experimental Procedures.''
The complex also catalyzes TNP-ATP hydrolysis under the single-site
hydrolysis condition (Fig. 4B, closed
circles), but this hydrolysis is not accelerated by the chase
incubation for 5 s with the addition of 1 µM ATP (Fig. 4B, open circles). The lack of the chase
acceleration at 1 µM ATP for the
complex cannot be attributed to the
dissociation of the complex. As described above, nucleotide-induced
destabilization of the
complex did
not occur under this condition. In addition, Sato et al.(1995)
reported that the dissociation of the
complex into the
complexes
induced by ATP proceeds fairly slowly; at 1 mM ATP, only very
little dissociation was observed in 20 s, and it took about 100 s for
the complete dissociation. We measured the amounts of ATP and ADP in
the reaction tubes of the experiment of Fig. 4B after
termination of the chase reaction and confirmed that added ATP was
almost completely hydrolyzed in 5 s (data not shown). Thus, lack of
chase acceleration for the complex may not be due to inability of ATP
at 1 µM to interact with the complex, even though a
possibility remains that ATP preferentially binds and is hydrolyzed by
the complexes without bound TNP-ATP. To be sure, we measured the effect
of chase addition of 1 mM ATP, and as expected no acceleration
was observed (data not shown).
Different from the
complex, single-site hydrolysis
of TNP-ATP by mutant
complexes
proceeded more slowly than that by wild-type
complex (Fig. 4, D, F, and H, closed circles). The hydrolysis
was not accelerated by the chase addition of 1 µM ATP (Fig. 4, D, F, and H, open
circles). The rate of single-site hydrolysis of TNP-ATP by each of
mutant
complexes shows a parallel
relation with the nucleotide binding affinity of each isolated mutant
subunit to ATP (Table 1).
When the same experiments of
difference spectra induced by the step-wise addition of TNP-ADP as
shown for the wild-type complex (Fig. 3) were repeated for the
complexes containing mutant
subunits, very similar transition of the spectra with increasing
TNP-ADP were observed, that is,
subunit-type spectra (molar ratio
<
1.5) at first and then
subunit-type spectra (molar
ratio >
1.5) (data not shown). Therefore, in agreement with the
results of single-site catalysis, a high affinity site is generated on
one of the mutant
subunits when the mutant
subunits are
assembled into the
complex in
spite of the fact that the isolated mutant
subunits have only
very weak affinities to nucleotides (Odaka et al., 1994). For
the mutant
complexes, the magnitude
of difference spectra were very small, and clear transition as
described above was not observed (data not shown).
The
importance of the subunit in coupling of ATP hydrolysis and
proton translocation was pointed out a long time ago (Yoshida et
al., 1977b), and recent studies using cross-linking and
fluorescent probes have provided evidence for movement or large
conformational change in the
subunit during catalysis (Turina and
Capaldi, 1994; Aggeler et al., 1995). Because the
complex should have a structure with
a perfect 3-fold symmetry (
)and it contains essentially no
endogenously bound nucleotide, structural asymmetry of the
complex is caused solely by the
subunit. Crystal structure of bovine MF
has shown
that each
subunit has a different contact with a centrally
located
subunit and is in a different state in terms of
nucleotide occupancy: one occupied by AMPPNP, another by ADP, and the
third by none (empty) (Abrahams et al., 1994). Our results
indicate that the heterogeneous state of the catalytic sites and their
mutual communication are dependent on structural asymmetry brought by
the
subunit.