(Received for publication, March 23, 1995; and in revised form, July 3, 1995)
From the
F-type ATPase from the thermophilic Bacillus PS3,
TFF
, which was essentially free of bound
nucleotides after isolation and purification, was co-reconstituted into
liposomes with the light-driven proton pump bacteriorhodopsin. The time
course of the light-induced ATP synthesis was biphasic; an initial slow
phase accelerated to a final steady-state rate two to three times
faster. Adding ATP before initiating the reaction suppressed the slow
phase, suggesting that the state of occupancy of specific sites by ATP
regulated the synthetic activity of TF
F
.
Incubating the purified TF
F
with ADP and ATP
revealed one ADP and two ATP binding sites that were stable to gel
filtration. We analyzed the time courses of light-induced ATP synthesis
for the enzyme with different nucleotide content, after
co-reconstitution into liposomes with bacteriorhodopsin. The two ATP
sites were identified to have regulatory function. A complex containing
TF
F
ADP, 1:1, was co-reconstituted with
various quantities of ATP to obtain a range of molar ratios of
TF
F
ADP:ATP of between 1:0 and 1:1.7. It
was found that the initial rate of ATP synthesis increased with the
level of ATP bound to the enzyme. After binding one ATP, a stimulation
of ATP synthesis by a factor of 2 was observed. The second ATP site
also exhibited regulatory properties. It stimulated ATP synthesis but
to a much smaller extent; the stimulation did not exceed 20%. Binding
of the photoreactive analogues
2-azido-[
-
P]ADP and
2-azido-[
-
P]ATP to the
TF
F
and their effects on the rate of ATP
synthesis are described further. Importantly, after covalent labeling
of the enzyme, tryptic digestion, and high performance liquid
chromatography purification, the label was found associated with the
Y364-containing tryptic peptide in all cases.
Y364 is in the
region of conserved residues GXEHYXXA, which is in
the
subunit and known to be part of the noncatalytic site.
The proton-translocating ATPase from the thermophilic Bacillus PS3, TFF
, (
)is a
transmembrane protein that catalyzes ATP synthesis coupled with proton
flux across the membrane(1, 2) . It belongs to the
class of F-type ATPases that all have in common a membrane-embedded
F
part, which mediates transmembrane proton conduction, and
a hydrophilic F
part, which contains the nucleotide binding
sites (3, 4, 5) . The F
part is
composed of five subunits with the stoichiometry
regardless of the
organism(6, 7, 8) . F
has been
studied extensively with respect to the nucleotide binding sites, and
the structure at 2.8 Å resolution has been reported recently for
beef heart F
(9) . It is generally accepted that
there are six nucleotide binding sites, three of them being potentially
catalytic and the other three
noncatalytic(10, 11, 12) . In this context
the nucleotide sites of the detached TF
(13) , the
core complex(14) , as well as the isolated
and
subunits (15) of the thermophilic ATPase have been
characterized in some detail. Interestingly, the TF
complex
has no endogenously bound adenine nucleotides(16) , a
characteristic that distinguishes it from the F
of other
species that contain 1-4 endogenously bound adenine
nucleotides/enzyme (17, 18) . This is an important
fact that essentially simplifies the study of the nucleotide binding
sites so that TF
can be seen as a model system with respect
to the binding sites. However, despite all of this information little
is known about the binding sites in the intact TF
F
and more surprisingly nothing about their roles during the
synthesis of ATP.
To understand the exact functional and/or
mechanistic role of the nucleotide binding sites during ATP synthesis,
we have co-reconstituted the whole enzyme TFF
with the light-driven proton pump bacteriorhodopsin into
liposomes. Upon illumination, relatively high rates of ATP synthesis
between 200 and 700 nmol of ATP/mg of TF
F
/min
were observed. Although the rate of synthesis was limited by the
light-induced pH gradient attainable with bacteriorhodopsin, the
co-reconstituted system has various advantages. (i) It is chemically
well defined, i.e. only two purified enzymes and a defined
lipid composition. (ii) It is physically well defined, i.e. proteoliposomes are homogeneous in size and in protein
distribution and orientation. (iii) It provides a stable and constant
transmembrane electrochemical potential gradient for many hours.
In
this work we have used a previously described procedure (for review see (19) ) for co-reconstitution of TFF
with bacteriorhodopsin to analyze in more detail the kinetics of
ATP synthesis and the role of the nucleotide binding sites. Three tight
binding sites that were stable to gel filtration were identified and
characterized. The ATP binding sites were identified to be responsible
for an acceleration of ATP synthesis by a factor of 2-3,
demonstrating for the first time that enzyme-bound nucleotides directly
affect synthesis of ATP by intact F-type ATPase. This is in agreement
with previous reports on the soluble F
part(20, 21) . Use of the photoaffinity
analogues 2-N
[
-
P]ADP and
2-N
[
-
P] ATP allowed us to
identify the three binding sites all of which contained the
derivatization of
Y364, which is in the region of conserved
residues GXEHYXXA. This peptide is common in all
F-type ATPases and is located on a loop that reaches into the
noncatalytic site on the
subunit(9) .
Figure 7:
HPLC elution profiles of photolabeled
TFF
after trypsin treatment. Photolabeled
TF
F
was trypsin treated and separated by HPLC.
The radioactivity of the collected fractions is represented by bars. The gradient in percent of eluent B is
superimposed. A and C represent the separation with
the ion exchange column. In A the enzyme was incubated with
100 µM ADP prior to the incubation with 100
µM 2-N
[
-
P]ATP. In C the enzyme was incubated with 100 µM
2-N
[
-
P]ADP. B and D represent a reverse phase column. In B the fraction
from A collected at 58 min was applied. In D the
fraction from C collected at 42 min was
applied.
Figure 1:
Light-driven ATP synthesis
by bacteriorhodopsin-FF
proteoliposomes.
Proteoliposomes were reconstituted at 25 °C from
phospholipid-bacteriorhodopsin-TF
F
-Triton X-100
micellar solutions supplemented with 20 mM octyl glucoside
before detergent removal. Final concentrations: lipid (4 mg/ml),
bacteriorhodopsin (200 µg/ml), TF
F
(30
µg/ml), Triton X-100 (8 mg/ml) in a medium containing 50
mM Na
SO
, 50 mM
K
SO
, 25 mM KH
PO
(pH 7.3). CF
F
was co-reconstituted with
bacteriorhodopsin under similar experimental conditions except that
octyl glucoside was omitted. After detergent removal by successive
addition of Bio-Beads SM-2, proteoliposomes were resuspended in the
same medium, supplemented with 2 mM ADP, and preilluminated
for 15 min at 40 °C. At time zero ATP synthesis was initiated by
adding 2 mM MgSO
. Aliquots were analyzed as a
function of time for their ATP content using luciferin-luciferase
assay. Shown are time courses of ATP synthesis by TF
F
proteoliposomes in the light (trace a), in the presence
of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (trace b), and in the dark (trace c). The time course
of ATP synthesis by CF
F
-bacteriorhodopsin
proteoliposomes in the light is shown in trace
d.
Thus it appears that the biphasic behavior observed during
ATP synthesis by TFF
is an intrinsic property
of this ATPase. Another characteristic that in part distinguishes
TF
F
from F-type ATPases from other sources such
as chloroplasts and mitochondria is that it can be isolated without any
nucleotide endogenously bound to any of the six
sites/enzyme(16) . This prompted us to check whether the
acceleration occurring during ATP synthesis through
TF
F
could be related to saturation of some ATP
binding sites by newly synthesized ATP.
Figure 2:
Light-driven ATP synthesis by
bacteriorhodopsin-TFF
proteoliposomes in the
presence of exogenous ATP. Shown are the time courses of ATP synthesis
as a function of ATP added together with 2 mM ADP to initiate
the reaction.
, 2 mM ADP;
, 2 mM ADP, 3
µM ATP;
, 2 mM ADP, 7 µM ATP;
, 2 mM ADP, 10 µM ATP.
The
role of ATP in determining the kinetic behavior of ATP synthesis was
investigated further by analyzing the time courses of the light-induced
ATP synthesis in experimental conditions where newly synthesized ATP
was continuously removed from the reaction medium. In Fig. 3the
rate of ATP synthesis by bacteriorhodopsin-TFF
proteoliposomes was measured through the formation of NADPH in a
reaction medium containing glucose, hexokinase, glucose-6-phosphate
dehydrogenase, and NADP (closed symbols). Under these
conditions the rate of ATP synthesis was not accelerated as compared
with the control experiment in which glucose was omitted and the
synthesized ATP was measured by the luciferin-luciferase assay (open
symbols). Throughout the illumination period, the rate of ATP synthesis
remained low, whereas in the control experiment the biphasic behavior
was again observed with an acceleration by a factor of 2.5 when about
10 µM ATP had been accumulated in the medium. (
)The experiments depicted in Fig. 2and Fig. 3demonstrated that, depending upon the concentration of ATP
present in the reaction medium, TF
F
can exist
in two different states, slow and a fast, with respect to the rate of
ATP synthesis.
Figure 3:
Light-driven ATP synthesis by
bacteriorhodopsin-TFF
liposomes as measured
with or without the coupled assay (hexokinase/glucose-6-phosphate
dehydrogenase). Bacteriorhodopsin-TF
F
proteoliposomes were reconstituted as described in Fig. 1.
After reconstitution, they were resuspended in a medium supplemented
with 1 mM NADP, 2 units/ml hexokinase, 2 units/ml
glucose-6-phosphate dehydrogenase, and with or without 1 mM glucose. After preillumination for 15 min in the presence of 2
mM MgSO
, 2 mM ADP was added, and ATP
synthesis was measured as a function of time with luciferin-luciferase
(
) or with the coupled assay when glucose was present
(
).
Figure 4:
Rate of ATP synthesis by
bacteriorhodopsin-TFF
proteoliposomes as a
function of ADP or P
concentrations. Proteoliposomes were
reconstituted as described in Fig. 1(except for a
lipid/bacteriorhodopsin of 10:1 w/w) in a buffer containing 50 mM Na
SO
, 50 mM K
SO
, and 25 mM Pipes (pH 7.4).
After detergent removal, the proteoliposomes were resuspended in the
same buffer, supplemented with 2 mM MgSO
, and
1-30 mM potassium phosphate (pH 7.4). After
preillumination for 15 min, ATP synthesis was initiated by adding
12.5-5,000 µM ADP. Panel A, rates of ATP
synthesis as a function of ADP concentration. For all samples 25 mM phosphate buffer was used.
, initial rates of ATP synthesis
measured up to 10 min after the reaction was initiated.
,
steady-state rates of ATP synthesis measured 20 min after the reaction
was initiated. Panel B, rates of ATP synthesis as a function
of P
concentration. For all samples 2 mM ADP was
used.
, initial rates of ATP synthesis measured up to 10 min
after the reaction was initiated.
, steady-state rates of ATP
synthesis measured 20 min after the reaction was
initiated.
First we determined the nucleotide content of
our purified TFF
preparation. The amounts of
ADP and ATP endogenously bound were found to be 0.4 and 0.1 mol/mol of
TF
F
, respectively (see Table 1),
confirming previous reports on purified TF
which has been
isolated almost free of tightly bound nucleotides(35) . In a
second set of experiments, the purified solubilized
TF
F
preparation was incubated in the presence
of nucleotides. To mimic the experimental conditions used for measuring
light-induced ATP synthesis in proteoliposomes, TF
F
was first incubated for 15 min at 40 °C in the presence of
ADP and Mg
, followed by a second incubation in the
presence of various amounts of ATP.
Incubation of
TFF
in the presence of 100 µM ADP
and 1 mM Mg
, followed by gel filtration
through Sephadex columns equilibrated in the same medium but without
ADP, indicated that 1 mol of ADP was tightly bound per mol of enzyme.
Increasing the incubation time or the ADP concentration did not lead to
further ADP binding, and a 1:1 TF
F
ADP
complex was obtained. Preincubation of TF
F
as
above with ADP and Mg
, followed by a second
incubation in the presence of ATP, revealed, in addition to one tightly
bound ADP, about two tightly bound ATP. As shown in Fig. 5the
binding of ATP to TF
F
is biphasic with respect
to the ATP concentration in the second incubation medium. 100
µM ATP led to 1 mol of ATP/mol of
TF
F
, and at 1 mM ATP to about 1.7 mol
of ATP tightly bound per mol of enzyme. (
)Interestingly,
although the unbound ATP could be hydrolyzed during the incubation, the
TF
F
ADP
ATP complexes were found to
be stable during repeated gel filtration indicating, that the bound
ATPs were not hydrolyzed even in the presence of 1 mM
Mg
. These findings suggest the presence of distinct
nucleotide binding and nucleotide hydrolyzing sites (see also (35) ).
Figure 5:
Binding of ATP by
TFF
. Purified TF
F
was
first incubated for 15 min at 40 °C with 100 µM ADP
and subsequently with various amounts of ATP, ranging from 0.01 to 1
mM. After gel filtration through Sephadex columns, the
different samples were analyzed for their tightly bound ATP content.
The number of tightly bound ATP/enzyme molecule is plotted versus the initial ATP concentration in the incubation medium (see
Footnote 4).
To investigate the effect of the tightly bound ATP,
TFF
, pretreated as described above, was
co-reconstituted with bacteriorhodopsin, and the resulting rates of
light-induced ATP synthesis were measured. ATP synthesis was started by
adding 2 mM ADP simultaneously with illumination of the
proteoliposomes. After a time lag of about 2 min, allowing the
generation of a stable transmembrane pH gradient, the rate of ATP
synthesis was monitored over a period of 10 min. Fig. 6gives
the rates of ATP synthesis as a function of ATP tightly bound to
TF
F
. The rates of ATP synthesis increased from
58 nmol of ATP/mg/min when no ATP was tightly bound to 100 nmol of
ATP/mg/min when 1 ATP was tightly bound to the
TF
F
. A further increase in tightly bound ATP
produced a small stimulation of ATP synthesis of 10-20%.
Figure 6:
Correlation between ATP tightly bound to
TFF
and stimulation of ATP synthesis by
bacteriorhodopsin-TF
F
proteoliposomes. Purified
TF
F
preparations treated as described in Fig. 5were recovered after gel filtration and contained
0.08-1.7 mol of ATP/mol of TF
F
. The
different treated enzymes were co-reconstituted with bacteriorhodopsin
as described in Fig. 1. ATP synthesis was then initiated
directly with light in the presence of 2 mM ADP, and aliquots
were analyzed for their ATP content every 3 min for 15 min. The figure
gives the rate of light-induced ATP synthesis as a function of ATP
tightly bound to
TF
F
.
Another important observation was that the stimulation through ATP was not observed when the proteoliposomes were preilluminated. Under conditions in which light-induced ATP synthesis was started by adding ADP after a 15-min preillumination of proteoliposomes, the stimulation was no longer observed, and ATP synthesis proceeded with a rate of 58 nmol of ATP/mg/min (only after 20 min of illumination the rate accelerated by a factor of 2-3). This observation indicated that the tightly bound ATP may have been released or changed to another binding site under energized conditions.
Also important is that when
purified TFF
was incubated with ATP without
prior incubation with ADP, the same nucleotide to enzyme ratios were
found after gel filtration (i.e. 1 ADP and 1.7 ATP/enzyme).
However co-reconstitution of this treated enzyme into liposomes led to
a slow initial ATP synthesis rate of 58 nmol/mg/min. This observation
suggested that different sites were occupied depending upon the order
of incubation with ADP and ATP during the pretreatment conditions of
TF
F
.
First we analyzed in detail the stoichiometry of labeling
by the 2-azido analogues. To quantify the amount of 2-azido nucleotides
covalently bound to the enzyme we incubated the solubilized enzyme at
40 °C in the dark in a medium containing 100 µM 2-N[
-
P]ATP and
Mg
. After a 15-min incubation the
TF
F
-nucleotide complex was separated from
unbound labeled nucleotides by gel filtration through Sephadex columns,
irradiated, and analyzed for its nucleotide content. Under these
conditions we found 1.2 labeled nucleotides/enzyme, which were stable
to acid precipitation, i.e. covalently bound (Table 1,
fourth row). Using unlabeled nucleotides, Table 1(third row)
shows that 100 µM ATP led to 0.8 ADP and 1 ATP tightly
bound per enzyme. Taking into account the initial amount of tightly
bound nucleotides, which were 0.4 ADP and 0.1 ATP (Table 1, first
and second rows), it can be estimated that the ATP treatment led to an
additional 1.3 nucleotides, comprising 0.4 ADP and 0.9 ATP. In another
set of experiments we preincubated the enzyme with 100 µM ADP before the addition of 100 µM 2-N
[
-
P]ATP. After Sephadex
columns and subsequent irradiation we found 0.6 azido
nucleotides/enzyme which were stable to acid precipitation. In the
control 0.7 additional ATP was found (Table 1, fifth and sixth
rows).
Second we investigated the effects of covalent labeling of
TFF
by 2-azidonucleotides on the kinetics of
ATP synthesis after co-reconstitution of the labeled
TF
F
with bacteriorhodopsin. After treating with
100 µM ADP, 100 µM
2-N
[
-
P]ATP, and
photoirradiation before reconstitution, ATP synthesis was monophasic (Table 1, sixth row). Taking into account the inhibition through
covalent labeling (see below), this corresponds to the fast rate. Two
control experiments were performed under similar conditions except that
either the enzyme incubated with
2-N
[
-
P]ATP was not
photoirradiated before reconstitution (not shown) or
2-N
[
-
P]ATP was replaced by
unlabeled ATP. Under these conditions monophasic behavior was found to
correspond to the fast rate of ATP synthesis (Table 1, fifth
row). However, when TF
F
was pretreated with 100
µM 2-N
[
-
P]ATP
without prior incubation with ADP, ATP synthesis was monophasic with a
very slow rate (Table 1, third and fourth rows). These results
confirmed that the nucleotide pattern of TF
F
by
ATP was dependent upon the order of incubation with ADP and ATP.
When TFF
was incubated with
2-N
[
-
P]ADP and photoirradiated,
the light-induced ATP synthesis (although halved) exhibited a biphasic
behavior as in the control with ADP (Table 1, seventh and eighth
rows). It is unlikely that the acceleration in ATP synthesis is a
result of the release of inhibitory ADP as suggested for the
mitochondrial ATPase (20, 21, 36) since (i)
in mitochondria the ADP is released from a catalytic site whereas in
our case it is a noncatalytic site (see next paragraph), and (ii) the
ADP analogue is bound covalently.
It should be noted that in all of these experiments the rates of ATP synthesis with the covalently photolabeled enzyme were lowered as compared with the controls. This could be due to inhibition of fractions of enzyme (37) or to a chemical modification which is in turn unfavorable for catalysis(15, 38) .
To
identify the amino acid sequence of the ADP and ATP binding sites we
trypsin treated the labeled enzyme and purified the fragments by HPLC
using ion exchange and a reverse phase column. After preincubation with
100 µM ADP followed by 100 µM 2-N[
-
P]ATP and
photoirradiation, the ion exchange column revealed two peaks, an ADP
and an ATP peak (Fig. 7A). The ATP peak was collected
and applied to a reverse phase column. It resulted in a single peak
that eluted at 28% of eluent B (Fig. 7B). Incubating
with 100 µM
2-N
[
-
P]ADP followed by
photoirradiation resulted in a single ADP peak after the ion exchange (Fig. 7C). We collected this peak and applied it to a
reverse phase column. The resulting single peak eluted at the same
place as the ATP, at 28% of eluent B (Fig. 7C). Amino
acid sequencing of the fragment isolated by reverse phase HPLC gave, in
both cases, the same sequence, ALAPEIV. . . . This sequence was
identified in the
subunit (39) as 353-ALAPEIVGEEHYQVA-367
with the Tyr-364 as the labeled amino acid (see also (15) ).
Tyrosine 364 is contained in the sequence GXEHYXXA,
which is a highly conserved sequence in F-type ATPases from bovine
mitochondria, spinach chloroplast, and Escherichia coli(30) and is part of the noncatalytic or regulatory site.
The studies described in this paper represent an effort to
understand the mechanisms and particularly the complex patterns of
nucleotide binding on F-type ATPase involved during ATP synthesis. The
most interesting finding emerging from this report is that ATP
synthesis by the F-type ATPase from the thermophilic Bacillus PS3 is shown to be significantly accelerated upon ATP binding to
the protein. On a more general level, this is the first report for an
intact F-type ATPase effect on the regulation of the catalytic process, i.e. ATP synthesis, by an enzyme bound nucleotide. In earlier
reports regulatory effects were only described for the mitochondrial
F in hydrolysis(20, 21) .
TFF
possesses fundamental similarities to
other energy-transducing ATP synthases of F-type. In particular there
is an agreement that F
, the water-soluble catalytic moiety
of this protein, contains six nucleotide sites that fall in two
categories: (i) three binding sites associated with a regulatory
function, and (ii) three catalytic sites. Despite a large number of
valuable reported studies on their binding properties as well as on
their functional and structural roles, a comprehensive classification
of the six sites is still lacking. There is now agreement about their
exact location; the catalytic sites are on the
subunits and the
noncatalytic on the
subunits. Although the structure is well
known, i.e. the
Y from the conserved sequence
GXEHYXXA is on a loop that reaches into the
subunit to the noncatalytic binding site(9) , the roles of the
nucleotides on the
subunits remain
unknown(10, 11, 40) . It should be stressed
that most investigations have been made with the isolated
F
, i.e. during hydrolysis of Mg-ATP, and only few
with the holoenzyme, although it is of primary importance since only
the TF
F
is capable of ATP synthesis, which is
the normal physiological response.
The use of
bacteriorhodopsin-TFF
co-reconstituted
proteoliposomes was very advantageous as it allowed investigation of
the mechanism of ATP synthesis and regulation by enzyme-bound
nucleotides. They are powerful tools for analyzing detailed mechanisms
inaccessible to study in complex native membranes and allow synthesis
through repeated enzyme cycles over a long time of experimental
observation. For comparison it should be pointed out that using
artificially imposed
]µ
transitions (
pH/
jumps), the reaction is terminated
within a few seconds(41) , rendering detailed kinetic
evaluations difficult. The only limitation of the co-reconstituted
systems is related to the small
]µ
generated by
bacteriorhodopsin which limits the turnover rate of the
H
-ATP synthase. However, for the purpose of the work
presented here, this limitation is an advantage since it allows a
slowdown in the process of ATP synthesis making the study of its
kinetic behavior easier.
It is clear from this study that
TFF
is subject to regulation during ATP
synthesis which depends on the state of occupancy of some specific
sites by ATP. The first experimental evidence came from the observation
that the rate of ATP synthesis by TF
F
was
dependent upon the concentration of ATP in the reaction medium. When
purified TF
F
was co-reconstituted into
proteoliposomes with bacteriorhodopsin, and a transmembrane pH gradient
had been established, ATP synthesis proceeded in two phases: an initial
slow one that gradually accelerated to a final steady-state rate. The
steady-state rate was about two to three times higher than the initial
slow rate and commenced when approximately 10 µM newly
synthesized ATP had accumulated in the assay medium. Continuous removal
of synthesized ATP led to a slow monophasic ATP synthesis (Fig. 3). Conversely, adding exogenous ATP before initiating the
reaction decreased the transition time between the slow and the rapid
phases. In the presence of 10 µM exogenous added ATP, ATP
synthesis proceeded only at the rapid steady-state rate (Fig. 2). It was further shown that the acceleration in ATP
synthesis did not influence the affinity for ADP and P
(Fig. 4). These observations indicated that
TF
F
contained specific ATP binding sites which,
once occupied, allowed ATP synthesis to proceed at a high rate.
Little is known about the nucleotide binding sites on
TFF
since the nucleotide binding properties are
investigated mainly in the disrupted TF
moiety and the
isolated subunits. There is a general agreement that
TF
F
as well as TF
can be isolated
and purified nearly free of nucleotides(35) ; however, the
results concerning adenine nucleotide binding to the TF
are
confusing. Using circular dichroism, Ohta and co-workers (35) first reported that in the presence of
Mg
, TF
F
contained three ADP
or alternatively three ATP sites stable to gel filtration and located
on both
and
subunits. Yoshida and Allison(13) ,
using [
H]ADP, described only one ADP site stable
to gel filtration which was located on the
subunit. Using
3`-O-(4-benzoyl)benzoyl ADP, Bar-Zvi et al.(42) demonstrated inhibition of the rate of ATP hydrolysis
by covalent binding to TF
; complete inactivation occurred
upon binding of 2 mol of Bz-ADP/mol TF
, and the label was
found to bind exclusively to the
subunit. It has also been
reported recently that there is a 95% inhibition of TF
ATPase activity in the presence of
2-N
[
,
-
P]ATP and
Mg
(15) . On comparison between the labeling
on the TF
and
or
subunits it was proposed that
the catalytic sites of F
resided exclusively on
subunit, whereas noncatalytic sites were located mostly on
subunit and that the adenine ring of ADP or ATP interacted with side
chains contributed by
subunits. Furthermore the literature (43, 44, 45) has analyzed the role of the
enzyme-bound nucleotides in terms of hydrolytic activity. Any
information about ATP binding sites on TF
F
and
their role in ATP synthase activity is lacking.
The results
presented in this paper are a first insight into the characterization
of the ATP binding sites of the thermophilic
FF
-ATPase. TF
F
can be
isolated and purified nearly free of bound nucleotides. When ADP was
added to the isolated enzyme in the presence of Mg
, a
1:1 TF
F
ADP complex was formed which was
stable to gel filtration. Binding of ATP, after preincubation with ADP,
revealed two additional sites for ATP which did not dissociate upon gel
filtration. Analysis of the binding of ATP after ADP incubation
revealed that one site saturated at 0.1 mM ATP, whereas the
other saturated above 1 mM ATP (Fig. 5). Thus the main
finding of these data is that TF
F
contained, in
addition to one ADP binding site, two ATP binding sites that were
stable to gel filtration.
The role of these ATP sites in regulating
the ATP synthesis process was analyzed after reconstitution into
liposomes (Fig. 6). The initial rate of the incorporated
TFF
ADP
ATP 1:1:1 complex was
stimulated 2-3-fold over that of the
TF
F
ADP 1:1 complex, and the initial rate
of the TF
F
ADP
ATP 1:1:1.7 complex
was further stimulated by 10-20%.
To characterize further the
ATP binding sites of TFF
, we used the
photoreactive analogue
2-N
[
-
P]AT(D)P which fulfilled
conditions for successful photoaffinity labeling experiments. In
particular it is a real substrate (i.e. it is hydrolyzed), and
the affinities for the adenine nucleotides and their 2-azido analogues
are nearly identical for F-type ATPases(46, 47) . When
TF
F
was first incubated with 100 µM ADP, followed by a second incubation with 100 µM
2-N
[
-
P]ATP, we found 0.7
2-N
[
-
P]adenine
nucleotides/enzyme. In the control experiment the azido compound was
replaced by ATP, and two nucleotides, 1.2 ADP and 0.8 ATP, were tightly
bound (Table 1, fifth and sixth rows). This clearly indicated
that 2-N
[
-
P]ATP was mainly
incorporated in the ATP site. When co-reconstituted into liposomes with
bacteriorhodopsin, this covalently labeled enzyme led to fast
monophasic behavior. Since fixing the analogue to the ATP site was
shown to fix the regulatory function it could be concluded that the ATP
sites were regulatory. We have also used
2-N
[
-
P]ATP to identify the
amino acid sequence of the binding site. After photolabeling, tryptic
digestion, and ion exchange HPLC, the ATP peak was collected and
applied to a reverse phase HPLC. The amino acid sequence then gave the
information that
Y364 was labeled. This site is in the conserved
region which is commonly named noncatalytic or regulatory(48) .
Finally our data provide some information about the ADP site in the
TFF
complex. The observation that after
preincubation with ADP
2-N
[
-
P]ATP was found mainly on
the ATP sites strongly suggested that the hydrolyzing
2-N
[
-
P]ATP did not chase the
ADP from the ADP site, i.e. the ADP site, which is stable to
gel filtration, is not part of the catalytic (hydrolytic) cycle. This
was clearly confirmed by the identification of the amino acid labeled
by 2-N
[
-
P]ADP which was
Y364. This can make a difference to the TF
since
Yoshida and Allison (13) provided evidence that on the ADP
site, stable to gel filtration, enzyme-bound ADP was converted to bound
ATP, which would point toward a catalytic site. However, the
experiments were performed in the presence of dimethyl sulfoxide, i.e. under extreme conditions. Thus an important feature of
this work is that it demonstrates that the three nucleotides that bound
tightly to TF
F
are noncatalytic, binding to one
ADP and two ATP sites.
In addition to this, several interesting
observations are derived from this study. (i) ATP bound to its sites
did not hydrolyze, suggesting that these sites are different from the
sites for ATP hydrolysis. (ii) In the absence of exogenous ADP, the ATP
sites are not stable to an electrochemical H gradient.
Preillumination of the proteoliposomes with the
TF
F
ADP
ATP complex in the absence of
exogenous ATP results in a biphasic kinetics of ATP synthesis, as
opposed to a monophasic fast kinetic observed when ATP synthesis was
started by illumination in the presence of exogenous ADP. A possible
explanation could be that in the presence of an electrochemical proton
gradient, tightly bound ATP is released and/or shifted to another site.
(iii) It was also observed that first binding ADP followed by ATP
binding is essential to gain a direct acceleration of ATP synthesis to
the fast phase. TF
F
preparations treated with
ATP without prior ADP incubation showed the same tightly bound
nucleotide pattern (i.e. a 1:1:2
TF
F
ADP
ATP complex) but led to
biphasic ATP synthesis. This can be interpreted assuming the existence
of a first and second site both able to bind ADP as well as ATP.
However to gain rapid ATP synthesis the nucleotides must each be bound
to a specific site. (iv) The observation that the 1:1
TF
F
2-N
[
-
P]ADP
complex in which 2-N
[
-
P]ADP was
covalently bound, exhibited the same biphasic kinetic behavior as the
TF
F
:ADP complex implies that the 2-3-fold
acceleration in ATP synthesis cannot be related to the release of
inhibitory ADP by newly synthesized ATP. (v) The ATP sites are true
regulatory sites in the sense that they are able to regulate the rate
of ATP synthesis.
In conclusion our studies allowed us to gain
further insight into the function of nucleotide binding sites during
ATP synthesis through TFF
. Other than the
various reports about nucleotide regulation of ATP hydrolysis of the
disrupted enzyme, i.e. the F
part (47, 49, 50) and nucleotides that play a role
in the activation(51) , this is the first report on the
regulation of the catalytic process, i.e. acceleration of ATP
synthesis by enzyme-bound nucleotide for an F-type ATPase. The fact
that the significant regulation of ATP synthesis by such sites has
never been reported with F-type ATPases from other sources may be
related to the fundamental difference between TF
F
and F
F
from other sources, which is that
the former can be isolated and purified free of bound nucleotides. For
instance, one can speculate that in the case of CF
F
from spinach chloroplast, which contains 1 ADP and 1 ATP tightly
bound after isolation and purification (12, 29) , only
the fast rate of ATP synthesis can be observed due to the fixed
nucleotides.