(Received for publication, September 29, 1995; and in revised form, November 14, 1995)
From the
It had previously been suggested that V hydrolysis rate of
2`,3`-O-(2,4,6-trinitrophenyl)adenosine 5`-triphosphate
(TNP-ATP) by F
-ATPase required filling of only two
catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S.
(1981) J. Biol. Chem. 256, 3718-3727), whereas recently
it was shown that V
rate of ATP hydrolysis
requires that all three catalytic sites are filled (Weber, J.,
Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E.(1993) J. Biol. Chem. 268, 20126-20133). To resolve this
apparent discrepancy, we measured equilibrium binding and hydrolysis of
MgTNP-ATP under identical conditions, using
Y331W mutant Escherichia coli F
-ATPase, in which the
genetically engineered tryptophan provides a direct fluorescent probe
of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at V
rate did require filling of all three
catalytic sites, but in contrast to the situation with MgATP,
``bisite hydrolysis'' of MgTNP-ATP amounted to a substantial
fraction (
40%) of V
.
Binding of
MgTNP-ATP to the three catalytic sites showed strong binding
cooperativity (K < 1 nM, K
= 23 nM, K
= 1.4 µM). Free
TNP-ATP (i.e. in presence of EDTA) bound to all three
catalytic sites with lower affinity but was not hydrolyzed. These data
emphasize that the presence of Mg
is critical for
cooperativity of substrate binding, formation of the very high affinity
first catalytic site, and hydrolytic activity in F
-ATPases
and that these three properties are strongly correlated.
ATP synthesis by oxidative phosphorylation is catalyzed by ATP
synthase. The F sector of this enzyme contains three
catalytic nucleotide binding sites, located on the three
-subunits
(Senior, 1988; Fillingame, 1990; Allison et al., 1992; Capaldi et al., 1994; Abrahams et al., 1994). F
may be isolated in soluble form; it is an active ATPase
(F
-ATPase), which has proved valuable for studies of
catalytic mechanism.
Since their introduction (Hiratsuka and Uchida,
1973), the trinitrophenyl (TNP) ()derivatives of adenine
nucleotides have been widely used to characterize nucleotide binding
sites of proteins and enzymes. These analogs have the advantages that
they are fluorescent and often bind with much higher affinity than the
natural nucleotides. Grubmeyer and Penefsky (1981a, 1981b) showed that
mitochondrial F
hydrolyzed MgTNP-ATP with K
1000 times lower and V
600 times lower than for MgATP. Importantly, these workers
demonstrated that MgTNP-ATP was hydrolyzed by (at least) two catalytic
sites on the enzyme and further that there was strong positive
catalytic cooperativity between catalytic sites, such that hydrolysis
of MgTNP-ATP pre-bound at a single site per F
was greatly
accelerated in presence of excess nucleotide sufficient to fill an
additional catalytic site(s) per enzyme molecule. Later, the same
cooperative behavior was detected with the natural substrate MgATP,
with acceleration factors of 10
-10
on
going from ``unisite'' to ``multisite'' catalysis,
not only for the mitochondrial enzyme (Cross et al., 1982) but
also for F
from other sources (Senior, 1988; Penefsky and
Cross, 1991).
A major question regarding the catalytic mechanism of
F has centered on whether occupation of two catalytic sites
by substrate is sufficient to achieve V
in
steady-state catalysis or whether occupation of all three sites is
required. Recently, the development of a fluorescent probe in the form
of a tryptophan residue specifically inserted into the catalytic sites,
which directly monitors the degree of occupancy of the sites by
nucleotide, has allowed us to answer this question. Using
Y331W
mutant Escherichia coli F
, we measured in parallel
experiments both MgATPase activity and degree of occupancy of the
catalytic sites as a function of MgATP concentration. A single K
value was found adequate to describe
the concentration dependence of MgATP hydrolysis, and this K
value was very similar to K
, the dissociation constant for binding
of MgATP to the third catalytic site (Weber et al., 1993).
Thus, steady-state hydrolysis of MgATP at physiological rate requires
that all three catalytic sites are filled with substrate. Filling of
only two sites was seen to generate at most a low and non-physiological
activity.
In contrast, for hydrolysis of MgTNP-ATP by mitochondrial
F, it had been suggested previously that V
was reached upon occupation of only two of the three catalytic
sites (Grubmeyer and Penefsky, 1981b). If this were the case, it would
imply that the enzyme utilizes all three catalytic sites for ATP
hydrolysis but only two sites for TNP-ATP hydrolysis. Considering that
TNP-nucleotides are probably the most frequently used fluorescent
analogs in studies of F
-ATPases, this would constitute a
serious discrepancy. The fact that V
for
MgTNP-ATP hydrolysis is very low might be taken as consistent with the
idea that MgTNP-ATP hydrolysis actually does occur by
``bisite'' catalysis, and indeed this low level of activity
might indicate the general order of magnitude for bisite activity with
other substrates. However, evidence from studies of isolated
-subunit (Rao et al., 1988) and a catalytic site peptide
fragment (Garboczi et al., 1988) suggests that each catalytic
site is potentially capable of binding MgTNP-ATP and therefore that in
intact F
all three catalytic sites would be expected to
bind MgTNP-ATP. Furthermore, intact F
was found to bind
three MgTNP-ADP (mol/mol) at saturation (Grubmeyer and Penefsky, 1981a;
Tiedge and Schäfer, 1986).
With the availability
of the Y331W mutant E. coli F
we are now able
to measure MgTNP-ATP binding to catalytic sites directly. In the study
presented here, we establish the relationship between catalytic site
occupancy and MgTNP-ATP hydrolytic activity, and we report the K
values for binding of MgTNP-ATP and
free TNP-ATP to F
catalytic sites.
Wild-type and Y331W mutant F
were prepared
from strains SWM1 (Rao et al., 1988) and pSWM4/JP17 (Weber et al., 1993), respectively, as described (Weber et
al., 1992). Before use, F
was equilibrated in 50
mM Tris/H
SO
, pH 8.0, by passing
100-µl aliquots consecutively through two 1-ml Sephadex G-50
centrifuge columns; this treatment reduced the amount of nucleotide
bound to catalytic sites to
0.2 mol/mol F
, as judged
from the fluorescence signal of
Y331W F
. Protein
concentration of F
solutions was determined using the
Bio-Rad protein assay (Bradford, 1976). The molecular mass of F
was taken as 382,000 Da (Senior and Wise, 1983). The F
concentration was 35-50 nM in all experiments
unless stated otherwise.
TNP-ATP was purchased from Molecular
Probes, Inc. (type T-7602, tri-sodium salt, supplied as 5 mg/ml
solution in 0.1 M Tris, pH 9, purity 96% by high pressure
liquid chromatography according to supplier) and was stored at
-20 °C. Purity of this material was checked by thin layer
chromatography as described by Grubmeyer and Penefsky (1981a) and
showed a single spot with mobility equal to that of TNP-ATP. Phosphate
analysis was carried out by the method of Taussky and Shorr(1953) after
complete hydrolysis by calf intestinal alkaline phosphatase or in 12 M HSO
and gave values of 3.13 and 3.02
mol P
per mol of TNP, respectively. Before hydrolysis, the
P
content was 0.02 mol/mol TNP. Concentration
determinations for TNP-ATP were based on an extinction coefficient of
26,400 M
cm
at 408 nm
(Hiratsuka and Uchida, 1973). All experiments were performed at 23
°C in buffer containing 50 mM Tris/H
SO
, pH 8.0, with further additions
as indicated. For measurements of hydrolytic activity, TNP-ATP (or ATP)
and MgSO
were added in a concentration ratio of 2.5 to 1.
MgTNP-ATP (and MgATP) concentrations were calculated according to
Fabiato and Fabiato(1979), assuming that the TNP moiety did not affect
Mg
complexation. Hydrolysis activities were
calculated from the amount of P
liberated; P
was determined by a very sensitive colorimetric assay (van
Veldhoven and Mannaerts, 1987).
Fluorescence experiments were
performed as described in Weber et al.(1993). For MgTNP-ATP
binding experiments, the buffer contained 50 mM Tris/HSO
, pH 8.0, with nucleotide and
MgSO
present in ratio of 2.5 to 1. For measurements of
binding of TNP-ATP in the absence of Mg
, the buffer
contained 0.5 mM EDTA instead of MgSO
. It should
be emphasized that the signal used to measure catalytic site MgTNP-ATP
binding throughout this study was the tryptophan fluorescence of
residue
W331. Even at the lowest concentrations used, binding of
MgTNP-ATP was complete in less than 30 s; under these conditions, less
than 5% of the analog was hydrolyzed. Parallel titrations of wild-type
enzyme with MgTNP-ATP were used to correct for inner filter effects
and/or resonance energy transfer from tryptophan residues other than
W331. Energy transfer from
W331 in one catalytic nucleotide
binding site to MgTNP-ATP bound in another catalytic site, which might
be considered a possible error source, is highly unlikely, as the
critical transfer distance for the donor/acceptor pair
tryptophan/TNP-ATP (
23 Å, Gryczynski et al.(1989))
is much smaller than the distance between catalytic sites (48 Å
as calculated from the x-ray structure, Abrahams et
al.(1994)). Enzyme prepared as described above contains
noncatalytic sites essentially filled with endogenous adenine
nucleotide. (
)In previous work with native E. coli F
, we showed that under the conditions used here for
fluorescence measurements in the presence of Mg
, no
release of noncatalytic site-bound nucleotide occurred over a period of
3 h (Weber and Senior, 1995). To test whether the presence of EDTA
induced release of nucleotide from noncatalytic sites, native F
was incubated in 50 mM Tris-SO
, pH 8.0, 5
mM EDTA at 23 °C, and nucleotide release was followed as
described by Weber and Senior(1995) (the presence of EDTA prevents
rebinding of released nucleotide, which is
Mg
-dependent). The calculated t
for release of 1 mol of noncatalytic site nucleotide
per mol of F
was 150 min. The fluorescence titrations were
performed in 10-fold lower concentration of EDTA (above), and the
signal was complete in
15 min. Therefore, no significant occupation
of noncatalytic sites by TNP-ATP could occur during the time courses of
the fluorescence experiments, and significant energy transfer between
W331 and noncatalytic site-bound TNP-nucleotide would not occur.
Fig. 1shows hydrolysis of MgTNP-ATP by wild-type E. coli F as a function of substrate
concentration. At saturation, the MgTNP-ATPase activity was 0.16
units/mg, which is 1.4% of the MgATPase activity under the same
conditions (23 °C, pH 8.0). The dashed line in Fig. 1is a fit to a model assuming Michaelis-Menten kinetics
with a single K
. However, it is evident
that the fit could be improved significantly using a model with two K
values (Fig. 1, solid
line). The calculated K
and V
values obtained using both models are given in Table 1. One point that is evident from these data is that E.
coli F
hydrolyzes MgTNP-ATP relatively better than
does mitochondrial F
, e.g. the ratio V
(MgTNP-ATP)/V
(MgATP) is
about 10-fold higher in E. coli F
as compared to
mitochondrial F
.
Figure 1:
Hydrolysis
of MgTNP-ATP by wild-type E. coli F-ATPase.
Hydrolysis of MgTNP-ATP was measured at 23 °C and pH 8.0 as
described under ``Materials and Methods.'' The dashed
line is a fit to the Michaelis-Menten equation assuming a single K
value; the solid line is a fit
assuming two K
values. Each data point (open circles) represents the average of at least duplicate
experiments.
In previous work we have used the
Y331W mutant E. coli F
extensively to
characterize catalytic site nucleotide-binding parameters (see
Introduction). It should be noted that this enzyme has properties
similar to wild type in both ATP hydrolysis and synthesis. Here, we
found that the enzymatic characteristics of
Y331W mutant F
with MgTNP-ATP as substrate (Fig. 2, open
circles) were also very similar to those of the wild-type enzyme.
At saturation, V
(MgTNP-ATP) (0.09 units/mg) was
1.5% of V
(MgATP). The dashed line in Fig. 2represents a model with a single K
. The fit could be improved considerably
by using a model with two K
values (Fig. 2, lower solid line). The calculated K
and V
values are
given in Table 1. It is evident from Table 1that in both
wild-type and mutant enzymes, for the model assuming two K
values the hydrolytic mode represented
by the lower K
value contributed
40%
of the total activity.
Figure 2:
Binding and hydrolysis of MgTNP-ATP by
Y331W mutant E. coli F
-ATPase. Hydrolysis of
MgTNP-ATP was assayed at 23 °C and pH 8.0 as described under
``Materials and Methods.'' The dashed line is a fit
to the Michaelis-Menten equation assuming a single K
value; the lower solid line is a fit assuming two K
values. Each data point (open
circles) represents the average of at least duplicate experiments.
Binding of MgTNP-ATP (filled circles) was measured under the
same conditions as hydrolysis, using the fluorescence of the
W331
residue as signal (see ``Materials and Methods''). Each data
point represents the average of at least duplicate experiments. The upper solid line is a fit to the binding data assuming a model
with a different K
value for each of
three binding sites.
Binding of MgTNP-ATP to Y331W mutant
F
catalytic sites was measured using fluorescence of the
genetically engineered tryptophan residue as signal. The results of
titration experiments with MgTNP-ATP are shown in Fig. 2(filled circles). At
10 µM MgTNP-ATP, the fluorescence of residue
W331 was completely
quenched, indicating that all three catalytic sites were filled.
Analysis of the MgTNP-ATP binding parameters using a non-linear
least-squares fit gave the following: site 1, K
1 nM; site 2, K
= 23 nM; site 3, K
= 1.39 µM. Compared to MgATP (Weber et al., 1993, 1994a), binding of MgTNP-ATP was therefore
20-30-fold tighter, but the same pattern was evident, i.e. binding to three sites with widely different affinity at each
site.
The agreement between K and K
on the one hand and K
and K
on the other is remarkable (Table 1). The results therefore
show that only enzyme molecules that have all three catalytic sites
filled with MgTNP-ATP are able to hydrolyze it at maximum rate.
However, it is apparent also that F
molecules that have
just two catalytic sites occupied by substrate do have significant
MgTNP-ATPase activity. This point is further examined in Fig. 3,
where the MgTNP-ATPase activity (filled circles) is plotted versus the fraction of catalytic binding sites occupied by
MgTNP-ATP. This latter parameter is of course an average for all the
enzyme molecules in the population. The solid line in Fig. 3shows the calculated activity expected if only enzyme
molecules with three substrate-filled sites are catalytically active (i.e. bisite activity = zero). The dashed line in Fig. 3shows the calculated activity expected if enzyme
molecules with two sites filled show 38% of the activity exhibited by
enzyme molecules that have all three sites filled. It is clear from Fig. 3that a model that ascribes partial (38%) activity to
enzyme molecules with two substrate-occupied sites results in a good
fit to the actual hydrolysis data.
Figure 3:
Estimation of the extent of bisite
hydrolysis of MgTNP-ATP by Y331W mutant E. coli F
-ATPase. Data for MgTNP-ATP hydrolysis (from Fig. 2) were plotted against the fraction of catalytic sites
occupied (solid circles). The latter parameter was calculated
from the binding data of Fig. 2. The solid line is the
calculated hydrolysis activity expected if bisite activity equals zero;
the dashed line is the calculated hydrolysis expected if
bisite activity equals 38% of V
(note: bisite
activity is defined as (V
We had previously shown that, in
absence of Mg, ATP bound to all three catalytic sites
of
Y331W mutant F
with the same affinity (K
= 76 µM) (Weber et al., 1994a). Binding of TNP-ATP was studied here under the
same conditions. As can be seen from Fig. 4, TNP-ATP also filled
all three catalytic sites in absence of Mg
. A model
assuming n identical, independent binding sites gave a
reasonable fit (Fig. 4, solid line) with calculated K
= 4.1 µM at 2.8
sites. However, a better fit was obtained using a model with three
different independent binding sites, with K
= 1.3 µM, K
= 4.1 µM, and K
= 32 µM (Fig. 4, dashed
line).
Figure 4:
Binding of TNP-ATP to Y331W mutant E. coli F
-ATPase in the absence of
Mg
. Free TNP-ATP binding in absence of Mg
was measured as described under ``Materials and
Methods'' (open circles). The F
concentration
was 90 nM. The solid line is a fit assuming n independent and equivalent sites. The dashed line is a
fit assuming three sites of differing affinity (see text for
details).
We found that in absence of Mg,
TNP-ATP was not a hydrolysis substrate. After incubation of 400 nM
Y331W mutant F
with 100 µM TNP-ATP
in presence of 0.5 mM EDTA for 3 h at 23 °C, pH 8.0, the
amount of released P
was found to be below the detection
limit of the assay (100 pmol). From the data, we estimate that the
TNP-ATP hydrolysis rate in absence of Mg
is below
10
units/mg, i.e. <0.01% of V
(MgTNP-ATP). Similar data were seen previously
for ATP hydrolysis in absence of Mg
(Weber et
al., 1994a).
The major objective of this study was to determine whether
maximal rates of MgTNP-ATP hydrolysis by F-ATPase are
achieved when just two of the three catalytic sites on the enzyme are
occupied by substrate, as had been suggested previously (Grubmeyer and
Penefsky, 1981b), or whether maximal rates are achieved only when all
three catalytic sites are filled, as had been demonstrated to be the
case for MgATP hydrolysis (Weber et al., 1993, 1994a). The
results established that V
rates of MgTNP-ATP
hydrolysis are achieved only when all three catalytic sites are filled.
An important difference between MgTNP-ATP and MgATP hydrolysis was
found, however. In previous work we showed that, for MgATP hydrolysis,
the rate of ``bisite'' activity (i.e. the rate of
hydrolysis manifested by an enzyme molecule having just two catalytic
sites filled) was but a small fraction of V, if
it occurred at all (Weber et al., 1993). The previous data
allow an estimate that bisite hydrolysis of MgATP could range from zero
to
2% of V
. Here, we saw that bisite
hydrolysis of MgTNP-ATP amounted to a substantial fraction (38%) of V
. It may be noted that in absolute terms this
rate of bisite hydrolysis is very slow, and indeed that V
for MgTNP-ATP hydrolysis by E. coli F
was only 1.4% of V
(MgATP).
Nevertheless, the fact that there is real bisite hydrolysis of
MgTNP-ATP may well imply that the rate of bisite hydrolysis of MgATP is
greater than zero.
We wish to emphasize that our conclusion that E. coli F shows bisite hydrolysis of MgTNP-ATP is
based on both kinetic experiments showing the appearance of two K
values and on equilibrium binding data
taken under the same conditions and that it is the agreement between K
values and K
values that supports our conclusion. In our opinion, it
would be unjustified to conclude that bisite hydrolysis occurred on the
basis of kinetic data alone, and for this reason we believe that this
is the first real demonstration of bisite hydrolysis by an F
enzyme.
Previously, the association rate constants for binding
of MgATP at the first and third catalytic sites of E. coli F have been calculated, and both are close to 10
M
s
(Weber et
al., 1994b). However the rate constant for binding to site 2 is
not yet known. From the data presented here, we can calculate k
/K
values for
MgTNP-ATP hydrolysis by wild-type E. coli F
in
both bisite and trisite hydrolysis, which are, respectively, 1.9
10
M
s
and 0.76
10
M
s
. This indicates that the rate of binding of
substrate to site 2 was 1 order of magnitude faster than binding to
site 3. This is a significant finding in view of the fact that the
enzyme is required to have all three sites filled to achieve
physiological rates of MgATP hydrolysis. It means that, should
nucleotide happen to dissociate from site 2 during the catalytic cycle,
thus temporarily disabling the enzyme, it would tend to rebind very
rapidly and restore the enzyme molecule to the fully active state.
Dissociation of nucleotide from site 1 is much less likely to occur
because the dissociation rate constant at this site is extremely slow
(Senior, 1988; Penefsky and Cross, 1991).
The equilibrium binding
data reported here showed that generally TNP-ATP mirrored ATP in its
behavior and was therefore a good analog for catalytic site ATP
binding. MgTNP-ATP was bound with 20-30-fold higher affinity than
MgATP but showed the same pattern of three sites with different
affinities. The presence of Mg was seen to cause a
huge increase in affinity for TNP-ATP at catalytic site one, as was
previously seen to be the case with ATP (Weber et al., 1994a).
The K
for binding of MgTNP-ATP at
catalytic site 3 was of similar magnitude to that for MgTNP-ATP binding
to isolated
-subunit (Rao et al., 1988) and also for
binding of free TNP-ATP at each of the catalytic sites in absence of
Mg
. Similar behavior was noted previously with MgATP
(Weber et al., 1994a). Therefore, the data presented here
provide additional evidence to support our previous conclusion that the
presence of Mg
is critical for manifestation of
strong substrate binding cooperativity, formation of the very high
affinity site one, and presence of catalytic activity and that these
three properties are strongly correlated.
In previous studies of
binding of free ATP (in absence of Mg), we found that
all three catalytic sites bound the nucleotide with the same K
value, and thus it appeared that all
three sites were equivalent under these conditions (Weber et
al., 1994a). The new data reported here with free TNP-ATP suggest,
however, that the three sites showed somewhat different affinities
toward the analog, although not to anywhere near the extent seen with
MgTNP-ATP. Therefore, it is apparent that even in the absence of
Mg
, the catalytic sites of F
-ATPase show
some degree of apparent ``asymmetry,'' in agreement with
recent chemical modification experiments (Haughton and Capaldi, 1995).