From the
Soluble F
In the energy-transducing membranes of mitochondria,
chloroplasts, and the plasma membrane of bacteria, the
H
Soluble F
In the
Ca
Here, we
studied the factors that may be involved in the hydrolysis and/or
release of ATP and PP
All nonradioactive chemicals were from Sigma.
[
Spontaneous synthesis of ATP and PP
The
methodology to determine the amount of radioactive
[
In various
experiments, after radioactive ATP and PP
In confirmation of previous data (Tuena de Gómez-Puyou et al., 1993), soluble mitochondrial F
The whole reaction mixture was also
centrifuged over Centricon filters; this procedure effectively
separated the enzyme from the media under conditions in which F
As noted, one of the questions
addressed here was if a change in solvent composition brought about
partition of ATP and/or PP
The experiment of Fig. 1was an attempt to determine
the time required for release of synthesized PP
For a long time it has been known that arsenate competes
with P
It
was found that F
It is also
noted that dilution with aqueous buffer that contained 3 mM of
ATP, ADP, or ITP did not produce release of synthesized ATP from
F
Research that has spanned for more than 3 decades has yielded
important information on the subunit composition, kinetics, control,
and structure of the whole ATP synthase and soluble F
The second part of the whole catalytic cycle, i.e. the release of spontaneously formed pyrophosphate bonds
into the solvent that surrounds soluble F
On the other hand, this work shows that in
soluble F
Spontaneous
formation of medium PP
Regarding the question of why in F
Formation of medium ATP from medium P
F
In experiment A,
F
F
from heart mitochondria incubated in
mixtures that have Mg
, inorganic phosphate, and
dimethyl sulfoxide (40% (v/v)) catalyzes the spontaneous synthesis of
ATP and pyrophosphate (Tuena de Gómez-Puyou, M., Garca, J. J.,
and Gómez-Puyou, A.(1993) Biochemistry 32,
2213-2218). By filtration techniques, it was determined that
synthesized ATP and pyrophosphate are enzyme bound, albeit the affinity
for pyrophosphate was lower than that of ATP. After ATP and
pyrophosphate were formed in dimethyl sulfoxide mixtures, dilution with
aqueous buffer to a dimethyl sulfoxide concentration of 6.0% brought
about the partition of pyrophosphate into the media. This was evidenced
by filtration experiments as well as by the accessibility of
synthesized pyrophosphate to soluble inorganic pyrophosphatase. Release
of pyrophosphate induced by dilution occurred in less than 15 s. Under
conditions that produce release of pyrophosphate, no release of ATP was
observed; instead, ATP underwent hydrolysis. Studies on the effect of
arsenate on the synthesis and hydrolysis of ATP and PP
in
F
showed that hydrolysis of synthesized PP
at
its site of synthesis was slower than that of ATP. Thus, the question
of whether differences in the rates of hydrolysis accounted for the
dilution-induced release of PP
but not of ATP was
addressed. Synthesis and hydrolysis of ATP and pyrophosphate were
examined in preparations of soluble F
in complex with its
inhibitor protein; the complex had an ATPase activity about 100 times
lower than that of free F
. In mixtures that contained
dimethyl sulfoxide, the complex synthesized ATP and pyrophosphate at
nearly the same rates; upon dilution, hydrolysis of both compounds
occurred also at similar rates, yet only pyrophosphate was released.
The same phenomenon was observed in F
that had been
depleted of adenine nucleotides. Hence, dilution-induced release of
PP
was independent of the overall catalytic properties of
the enzyme or its content of adenine nucleotides. Since synthesis of
ATP occurs at the expense of the ADP that remains after depletion of
adenine nucleotides, it is likely that the failure of ATP to be
released is due to the high affinity that F
exhibits for
the synthesized ATP. Nevertheless, the results illustrate that a
complete catalytic cycle that starts with medium P
and ends
with medium pyrophosphate may be reproduced in soluble mitochondrial
F
.
-ATP synthase catalyzes the synthesis of medium ATP
from medium ADP and P
utilizing the energy of
electrochemical proton gradients derived from electron transport (for
reviews see Futai et al., 1989; Penefsky and Cross, 1991). The
same basic process occurs in the light-driven synthesis of PP
from P
by a membrane-bound pyrophosphatase in
chromatophores of Rhodospirillumrubrum (Baltscheffsky et al., 1966). The catalytic moiety for
ATP synthesis of the ATP synthase is F
; this may be
detached from the membrane as a soluble multisubunit protein. Soluble
F
can also be obtained (Gómez-Puyou et al.,
1986) in complex with the inhibitor protein of Pullman and
Monroy(1963). F
has six adenine nucleotide binding sites
(Penefsky and Cross, 1991); pyrophosphate may bind to some of these
sites (Issartel et al., 1987; Peinnequin et al.,
1992). It has also been shown that a synthesized peptide based on the
amino acid sequence of F
has the capacity to bind PP
(Garboczi et al., 1988).
placed
in media that contain Me
SO
(
)catalyzes the spontaneous synthesis of ATP from
medium P
and enzyme-bound ADP (Sakamoto and Tonomura, 1983;
Yoshida, 1983; Sakamoto, 1984; Gómez-Puyou et al.,
1986; Kandpal et al., 1987; Beharry and Bragg, 1991a, 1991b;
Beharry and Bragg, 1992). More recently, it was found that soluble
F
incubated in Me
SO also has the capacity to
form PP
(Tuena de Gómez-Puyou et al.,
1993). In consonance with the observation that ATP and PP
are formed spontaneously in the presence of Me
SO,
studies on the rate and equilibrium constants of the steps of the
catalytic cycle in F
showed that at the active center of
F
, the equilibrium constant for ATP hydrolysis is close to
one (Grubmeyer and Penefsky, 1981; Wood et al., 1987; Al-Shawi et al., 1990; Al-Shawi and Senior, 1988, 1992).
-ATPase of sarcoplasmic reticulum, it was observed
that in mixtures with Me
SO, P
may phosphorylate
the aspartate that lies at its catalytic site (de Meis et al.,
1980); this in turn may yield medium ATP upon transfer of the
phosphorylated enzyme to aqueous media that contain ADP. It is also
known that in presence of Me
SO, soluble inorganic
pyrophosphatase catalyzes synthesis of medium PP
(de Meis,
1984). The findings with the Ca
ATPase and soluble
inorganic pyrophosphatase illustrate that changes of the aqueous
environment result in formation of medium ATP and PP
. As
noted, the spontaneous synthesis of ATP in F
incubated with
Me
SO is well documented; however, the factors that control
its partition into the media have not been explored in sufficient
detail. The studies are of importance, since it has been proposed that
during oxidative phosphorylation, release of ATP into the media is an
energy-requiring step (Boyer et al., 1973; Grubmeyer et
al., 1982; Penefsky, 1985). Thus, studies on the release of
spontaneously formed ATP and/or PP
may be particularly
relevant as they may shed light on the mechanisms that operate in the
transformation of the energy of electrochemical H
gradients into that of medium ATP or PP
.
that had been formed spontaneously in
soluble mitochondrial F
after incubation in
Me
SO. The results show that in a Me
SO to water
transition, PP
is ejected into the media. Thus, synthesis
of medium PP
from medium P
was achieved in the
absence of electrochemical H
gradients. In contrast,
under a number of conditions the enzyme failed to release spontaneously
formed ATP. The results suggest that at least with PP
,
solvation is central in the events that lead to formation of
``high energy'' bonds in aqueous media.
P]Phosphate was from DuPont NEN; it was
purified as described elsewhere (de Meis, 1984). F
was
prepared from bovine heart submitochondrial particles as described
before (Tuena de Gómez-Puyou and Gómez-Puyou, 1977) and
stored at 4 °C in 50% ammonium sulfate that also contained 1
mM ATP and 1 mM EDTA, pH 8.0; F
in
complex with the inhibitor was purified from submitochondrial particles
whose ATPase was controlled by the inhibitor protein as described
elsewhere (Gómez-Puyou et al., 1986). Prior to the
experiments, free F
and F
-IP were centrifuged
through Sephadex G-50 columns equilibrated with 40 mM MOPS-KOH, pH 6.8 (Kasahara and Penefsky, 1978). F
depleted of adenine nucleotides was prepared according to Garret
and Penefsky, 1975) and used as described before (Tuena de
Gómez-Puyou et al., 1993). Protein was determined
according to Lowry et al.(1951) using bovine serum albumin as
standard.
was
determined as previously described (Tuena de Gómez-Puyou et
al., 1993). F
was incubated at concentrations that
ranged between 2.2 and 2.9 µM in 40 mM MOPS, 10
mM MgCl
, 2 mM
[
P]P
, and 40% Me
SO (by
volume), pH 6.8. The specific activity of
[
P]P
was around 2
10
cpm/nmol. Generally, the volume of the incubation mixture was 250
µl, except in experiments in which 250-µl aliquots were
withdrawn at various incubation times; it is referred to as standard
synthetic mixture. Variations in the composition of the mixture are
detailed under ``Results.'' Temperature was 30 °C, except
in the experiments with the F
-inhibitor protein complex, in
which the temperature was 25 °C. Synthesized radioactive ATP and
PP
was determined as previously described (Tuena de
Gómez-Puyou et al., 1993) with a few variations. The
reaction was arrested with 6.0% trichloroacetic acid (final
concentration). This was followed by addition of 500 µM nonradioactive ATP and PP
. To the mixture, 0.5 ml of
3.3% ammonium molybdate in 3.75 N H
SO
was added. Thereafter, 1 ml of water-saturated isobutanol/benzene
(1:1 by volume) was added followed by vigorous stirring. After phase
separation, the organic phase was removed. To the water phase, 20
µl of 20 mM nonradioactive P
was added and
extracted again with isobutanol/benzene. This step was repeated four
times, but in the last two extractions, 20 and 10 µl of 100 mM nonradioactive P
were added. After the last
extraction, most of the ammonium molybdate had been removed.
P]P
incorporated into ATP and
PP
was as follows. The extract obtained above was
neutralized to pH 7.0-7.5. Two equal aliquots of the mixture were
withdrawn. One received 1 unit of soluble yeast inorganic
pyrophosphatase and was incubated for 30 min. At this time, 0.5 ml of
ammonium molybdate in H
SO
was added to the two
aliquots and extracted three times with isobutanol/benzene. The
radioactivity of the water phase was determined. The difference in
radioactivity between the samples that had or had not received soluble
inorganic pyrophosphatase was considered to correspond to pyrophosphate
synthesized by F
. As shown before (Tuena de
Gómez-Puyou et al., 1993), radioactivity insensitive to
pyrophosphatase activity corresponds to
[
P]ATP. Blanks for every experimental
condition were made; these were reaction mixtures that had no
F
; they were treated as the experimental.
were formed by
F
, the synthetic mixture was either centrifuged (Kasahara
and Penefsky, 1978) through Sephadex columns equilibrated with the
synthetic mixture that contained nonradioactive P
and 30%
Me
SO or filtered through Centricon 10 filters (Amicon). In
other experiments, the synthetic reaction mixture was diluted with 6
volumes of all aqueous 40 mM MOPS buffer, pH 7.0. In this
case, the diluted mixture was filtered through Centricon filters, and
radioactive ATP and PP
were determined in the filtrate;
alternatively, after dilution, 6.0% trichloroacetic acid (final
concentration) was added at various times, and the extraction procedure
described above was followed to determine radioactive ATP and
PP
.
incubated
in media that contained P
, Mg
, and 40%
Me
SO catalyzed the spontaneous formation of ATP and
PP
(). Concerning the characteristics of ATP
and PP
synthesized by F
, it was relevant to
ascertain if the totality or only a portion of the ATP and PP
formed was enzyme bound. The mixture in which the enzyme had been
allowed to form ATP and PP
was passed through centrifuge
columns filled with Sephadex G-50. The ratio of ATP and PP
per enzyme was determined before and after centrifuge column
elution (). The amount of ATP bound per enzyme was of a
similar extent in the two fractions; hence, synthesized ATP was enzyme
bound. On the other hand, the amount of PP
per F
was lower in the eluate, which indicated that either a fraction
of the synthesized PP
was medium PP
or that a
portion of the enzyme-bound PP
was released during passage
through centrifuge columns.
was always in contact with the media. In the filtrate, no ATP or
PP
were detected (). Taken together, the
results indicated that synthesized ATP and PP
remained
bound to F
. However, the affinity of F
for
PP
was lower than that for ATP, since a fraction of bound
PP
was lost upon passage through Sephadex centrifuge
columns.
Effect of Dilution on ATP and PP
At concentrations of Me Bound to
F
SO below 10%
and concentrations of P
in the range of 2 mM,
synthesis of ATP and PP
is hardly detectable (Sakamoto and
Tonomura, 1983; Tuena de Gómez-Puyou et al., 1993).
Hence, the effect of diluting with aqueous buffer the 40%
Me
SO synthetic mixture in which soluble F
had
been allowed to form ATP and PP
was studied. In agreement
with previous data (Sakamoto and Tonomura, 1983; Gómez-Puyou et al., 1986), it was observed that dilution of the reaction
mixture with 6 volumes of aqueous buffer brought about the breakdown of
ATP (, Exp. A). In over ten experiments, after 1 h of
dilution, between 50 and 80% of the ATP was hydrolyzed. In contrast, in
the same number of experiments, it was observed that in 1 h after
dilution at least 80% of the synthesized PP
remained in the
mixture (, Exp. A).
from their site of synthesis
into the media. To this purpose, F
was allowed to form ATP
and PP
in the standard synthetic mixture. Thereafter, the
mixture was diluted and passed through Centricon filters; in the
filtrate, ATP and PP
were determined. (Exp.
B) shows that a substantial amount of radioactive PP
appeared in the filtrate. Thus, dilution brought about partition
of synthesized PP
into the aqueous media. However, only a
fraction of the total PP
(about 70%) appeared in the
filtrate.
from
F
after a Me
SO to water transition. F
that had been allowed to form PP
was diluted with
aqueous buffer that contained inorganic pyrophosphatase. Within 15 s,
almost the totality of the synthesized PP
disappeared from
the system. These results, together with the data obtained from the
filtration experiments that show that only a portion of the total
PP
formed is released by dilution, indicate that full
release of PP
took place in less than 15 s, provided the
equilibrium between enzyme-bound and free PP
is shifted by
hydrolysis of the latter.
Figure 1:
Release of synthesized PP
by dilution of the synthetic reaction mixture. F
(2.4
µM) was incubated for 4 h in the synthetic reaction
mixture. [
P]Phosphate uptake into ATP and
PP
was 0.07 and 0.38 nmol/nmol F
, respectively;
these values are 100% in the figure. At that time, the mixture was
diluted with 6 volumes of 40 mM MOPS, pH 7.0, that contained 2
units of soluble inorganic pyrophosphatase. At the times indicated,
aliquots were withdrawn for assay of ATP and
PP
.
In contrast to PP, no ATP was
detected in the Centricon filtrate. In this respect, it is noted that
filtration took between 45 and 60 min. Since ATP is gradually
hydrolyzed after dilution, it was considered that ATP may have been
released and hydrolyzed prior to its removal from the whole reaction
mixture. However, when the synthetic mixture was diluted with aqueous
buffer that contained hexokinase + glucose
(
)(activity in excess over that of the ATPase activity
of F
), there was disappearance of non-extractable
[
P]P
(see ``Materials and
Methods''). If ATP had been released, it would have been trapped
by hexokinase yielding nonextractable glucose 6-phosphate. Thus,
dilution did not cause release of ATP from F
from the
enzyme into media, albeit it brought about partition of PP
.
Hydrolysis of Synthesized ATP and PP
The different behavior that ATP and PP at Their
Site of Synthesis
exhibited in the aforementioned Me
SO to water
transition could be used to ascertain the factors that control their
release or hinder their partition into the media. At their site of
synthesis, ATP and PP
could follow two competing pathways:
hydrolysis or partition into the media. In principle, the pathway that
either of the two compounds can follow would depend on the relative
rates of the two reactions. Hence, it was necessary to ascertain the
relative rates of hydrolysis of the two compounds at their site of
synthesis. In this regard, it is noted that Kandpal et
al.(1987) and Al-Shawi and Senior(1988, 1992) showed that in
presence of Me
SO, synthesized ATP undergoes reversible
hydrolysis.
in oxidative phosphorylation, and it was described
that it may arsenylate ADP yielding an unstable analog of ATP (Crane
and Lipmann, 1953). Hence, arsenate was used to study whether ATP and
PP
undergo reversible hydrolysis at the catalytic site.
Prior to these studies, synthesis of ATP and PP
were
determined at various P
concentrations. Lower
concentrations were required for ATP than for PP
synthesis (Fig. 2). For example, substantial formation of ATP was observed
with 0.5 mM P
, a concentration at which PP
synthesis was not detected.
Figure 2:
Synthesis of ATP and PP at
various concentrations of phosphate. F
(2.2
µM) was incubated in the standard synthetic mixture,
except that it contained the indicated concentrations of P
.
Incubation time was 30 min. Open and closedsymbols indicate two separate experiments. Circles and triangles indicate the amount of
[
P]phosphate incorporated into ATP and
PP
, respectively, as mol/mol
F
.
With 2.0 mM P, 5 mM arsenate produced almost complete
inhibition of ATP and PP
synthesis (opentriangles in Fig. 3). The figure also shows that in
3 and 5 h of incubation, the level of synthesized ATP remained fairly
constant. However, when arsenate was added to the enzyme that had been
allowed to form ATP for 3 h (time zero in Fig. 3), there was a
progressive decrease in the level of radioactive ATP. This indicated
that at the catalytic site, synthesized ATP underwent reversible
hydrolysis and that arsenate diminished the rate of the forward
reaction; otherwise, the level of ATP that existed at the time of
arsenate addition would have remained constant.
Figure 3:
Effect of arsenate on ATP and PP synthesis and on previously synthesized ATP and PP
.
F
(2.2 µM) was incubated in the standard
conditions for synthesis without and with 5 mM arsenate for 3
h. At that time, the amounts of ATP and PP
were determined.
In the absence of arsenate, 0.1 and 0.49 nmol of
[
P]phosphate per nmol of F
had been
incorporated into ATP and PP
, respectively; these values
are 100% in the figure. The opentriangle indicates
the amount of ATP and PP
formed with 5 mM arsenate
added at the beginning of the experiment; in both cases, there was only
about 2% uptake. At the 3-h incubation period (time zero in the
experiment), 5 mM arsenate was added to F
that had
been incubated without arsenate. At the times shown, aliquots were
withdrawn to determine ATP and PP
(closedsquares and closedcircles,
respectively). The opensquare and opencircle indicate the amount of ATP and PP
,
respectively, in mixtures that did not receive
arsenate.
With PP,
different results were obtained (Fig. 3). In the absence of
arsenate, the amount of synthesized PP
increased in the
3-5-h incubation time (in all time curves we have carried out
with F
, synthesis of PP
continues for times
longer than those with ATP, i.e. synthesis of ATP levels off
at around 1 h, whereas that of PP
continues to increase
even after 6 h). The addition of arsenate to F
that had
been allowed to form PP
produced a rather slow decrease in
the pre-existing level of PP
. In fact, no effect of
arsenate on PP
levels was observed after 90 min of arsenate
addition; a moderate decrease was observed only after 2 h. The latter
indicates that under synthetic conditions, and similarly to ATP, there
is continuous synthesis and hydrolysis of PP
at the
catalytic center but that its rate of hydrolysis is slower than that of
ATP.
ATP and PP
The results with
arsenate raised the possibility that hindrances in hydrolysis of
PP Synthesized in Soluble F
Natural ATPase Inhibitor Protein Complex
at its site of synthesis could be related to its release
upon dilution, conversely that the higher rate of hydrolysis of
synthesized ATP could account for a predominance of the hydrolytic
pathway. To address the problem, a system in which hydrolysis of
PP
and ATP took place at equivalent rates was looked for.
To this purpose, we examined the process of synthesis of ATP and
PP
in soluble F
when in complex with the
natural ATPase inhibitor of Pullman and Monroy, 1963). The complex
(F
-IP) can be prepared from submitochondrial particles that
have their ATPase controlled by its inhibitor protein
(Gómez-Puyou et al., 1986); it exhibits a hydrolytic
activity about 100 times lower than that of free F
.
-IP has the capacity to synthesize ATP and
PP
at nearly equal rates (Fig. 4). When the synthetic
mixture was diluted, the two compounds were hydrolyzed; however, it is
relevant that the breakdown of ATP and PP
occurred at
rather similar velocities (Fig. 4). This was a fortunate
circumstance, since with F
-IP, the question of whether at
equivalent rates of hydrolysis and synthesized ATP and PP
followed the same or different pathways upon dilution could be
addressed.
Figure 4:
Synthesis of ATP and PP by
F
in complex with its inhibitor protein. The F
complex (2.6 µM) was incubated in the standard
synthetic mixture. At the times shown, aliquots were withdrawn for
assay of ATP and PP
. 100% in the figure represents nmol
[
P]P
taken up into ATP (0.19 nmol)
and PP
(0.09) per nmol of F
, respectively.
After 4 h of incubation, the synthetic mixture was diluted with 6
volumes of aqueous MOPS buffer, pH 7.4. Afterward, aliquots were again
withdrawn for assay of ATP and PP
. It has been described
that the F
-IP complex can be activated in prolonged
incubation times by relatively high temperatures and salts
(Beltrán et al., 1984). To avoid activation, the
experiments were carried out at a temperature of 25 °C. Hydrolysis
was also determined at the beginning and end of the experiments; these
values were 0.3 and 1.0 µmol/min/mg, respectively. Thus, the
complex did not undergo significant activation during the course of the
experiment.
F-IP was allowed to synthesize radioactive
ATP and PP
in standard incubation mixtures and was
subsequently diluted. The presence of ATP and PP
in the
media was examined by filtration and enzyme-trapping experiments (I). As evidenced by the appearance of PP
in
Centricon filtrates and its accessibility to inorganic pyrophosphatase,
PP
was released into the media. On the other hand, no
evidence that indicated release of ATP was observed.
-IP; also, it was observed that the presence of the
nucleotides did not affect the dilution-induced release of PP
(not shown).
Studies with F
As previously described, F Depleted of Adenine
Nucleotides
that has a
low content of adenine nucleotides (0.4 mol of ADP and less than 0.1
mol of ATP/mol F
) synthesizes ATP and PP
(Tuena
de Gómez-Puyou et al., 1993). Adenine
nucleotide-depleted F
was allowed to to synthesize 0.03 ATP
and 0.04 PP
per F
in mixtures with 30%
Me
SO in a 2-h incubation time (these conditions were used
to avoid inactivation of adenine nucleotide-depleted F
,
which is labile in mixtures with 40% Me
SO; see Tuena de
Gómez-Puyou et al., 1993). Upon dilution, only PP
was released. The results with this preparation are interesting
in the sense that they indicate 1) that the nucleotide content of
F
does not affect ATP or PP
release and 2) that
synthesis of ATP takes place with the ADP that remains in the enzyme
after adenine nucleotide depletion (in other words, with the ADP that
is bound to F
with the highest affinity).
.
Moreover, the crystallographic structure of soluble F
has
been described by two independent groups of researchers (Bianchet et al., 1991; Abrahams et al., 1994). Regardless of
these extensive studies, the basic question of how energy of
electrochemical H
gradients is transformed into high
energy pyrophosphate bonds in the medium has not yet been unveiled. In
this context, it is relevant that at the catalytic center of the ATP
synthase, the equilibrium constant of ATP hydrolysis, is close to one.
In consonance with these observations is that spontaneous synthesis of
ATP and PP
may take place in soluble F
,
provided the mixture contains a cosolvent that favors the partition of
P
into the catalytic site (Tuena de Gómez-Puyou et al., 1993). Thus, the part of the catalytic cycle that
leads to formation of enzyme-bound ATP has been reproduced in the
soluble enzyme.
, has proved more
elusive. In fact, the present work shows that under a number of
conditions and using F
with different nucleotide
composition and markedly different ATPase activities, no formation of
medium ATP was observed.
with different characteristics, formation of
medium PP
from medium P
could be readily
achieved. Synthesis of medium PP
involved two steps. The
first was the spontaneous formation of PP
at a catalytic
site in F
at the expense of medium P
in
presence of Me
SO. The second involved a transition from the
Me
SO media to a nearly all aqueous mixture; this resulted
in the ejection of PP
into the media. Hence, a whole
catalytic cycle that started with medium P
and ended with a
high energy phosphate bond in aqueous media could be reproduced in the
absence of electrochemical H
gradients.
from medium P
has been
observed before; soluble inorganic pyrophosphatase incubated in
Me
SO catalyzes the synthesis of medium PP
in
amounts larger than enzyme (de Meis, 1984). This was a consequence of a
change in the equilibrium constant of PP
hydrolysis, which
in Me
SO media has a less negative free energy of hydrolysis
than in all aqueous mixtures (de Meis, 1989). The presently described
synthesis of either enzyme bound or medium PP
was less than
one per F
. Thus, although F
has the catalytic
properties to synthesize and hydrolyze PP
, and the
conditions in Me
SO mixtures were thermodynamically
favorable for formation of medium PP
, the catalytic cycle
in F
stopped with PP
at the catalytic center.
In the light of the data with soluble inorganic pyrophosphatase, it may
be inferred that release of PP
from F
into the
media of synthetic mixtures is not hindered by energetic barriers but
rather by kinetic barriers. Of particular relevance is that in
F
, these barriers are overcome by exposing the enzyme to
more aqueous environment. In consequence, it would seem that solvation
of F
bears strongly on the magnitude of the kinetic
barriers that lead to release of synthesized PP
.
, PP
but
not ATP is released in a Me
SO to water transition, the
results with different F
preparations show that the
difference cannot be ascribed to different catalytic rates or occupancy
of adenine nucleotide binding sites. Hence, it is possible that release
of ATP and PP
involve different mechanisms. However, on the
assumption that solvation of F
also bears on ATP release,
it is likely that differences in partition between ATP and PP
are due to their different chemical structure, and thus they
would have different affinity for the catalytic site. The crystal
structure of F
reveals that the adenine group of the ATP
molecule interacts with various residues of the catalytic sites
(Abrahams et al., 1994). Since these interactions do not occur
with PP
, it is possible that the higher number of points
with which ATP interacts confers a higher stability of ATP at the
catalytic site. F
from Escherichia coli that has
been depleted of adenine nucleotides exhibits reversible synthesis of
ATP at the catalytic site with the same rate constants as native
F
(Senior et al., 1992). Thus, it is relevant that
in heart adenine nucleotide-depleted F
, ATP synthesis
occurred at the expense of the ADP that remained bound; this implies
that the ADP with the highest affinity was the species phosphorylated.
Hence, it is possible that at that site, F
continued to
exhibit a high affinity for the synthesized ATP. In fact, it has been
reported (Senior et al., 1992; Al-Shawi and Senior, 1992) that
at the catalytic site of F
from E. coli, the K
for Mg-ATP is 10
in
aqueous media.
and
ADP in the absence of ion gradients was first described with the
Ca
of the sarcoplasmic reticulum (de Meis et
al., 1980). Changes in solvent were instrumental in such synthesis
(formation of acyl-phosphate by medium P
in presence of
Me
SO and subsequent synthesis of ATP upon transfer of the
phosphorylated enzyme to aqueous media that contained ADP). Hence, and
particularly in the light of the solvent-induced release of PP
from F
, it is worthwhile to consider if solvation of
F
forms part of the overall mechanism that operates during
coupled electron transport in energy-transducing membranes.
Table: Distribution of synthesized ATP and PP between F
and medium
was incubated for 4 h at a concentration of 2.4 µM in the synthetic reaction media. It formed the amount of
[
P]ATP or PP
indicated. At that
time, an aliquot was passed through Sephadex G-50 centrifuge columns
equilibrated with the same mixture, except that it contained
nonradioactive P
, and the concentration of Me
SO
was 30%. In the eluate, protein and the amount of
[
P] incorporated into ATP and PP
was
determined. At the same time, an aliquot of the synthetic reaction
mixture was applied to Centricon filters, and the media separated from
the enzyme. No ATP or PP
were detected in the filtrate
(ND). Filtration through Centricon filters took 2 h.
Table: Effect of dilution
on synthesized ATP and PP
(2.4 µM) was allowed to synthesize ATP and
PP
for 4 h to the levels shown (time zero in the
experiment). At this time, the mixture was diluted with 6 volumes of
totally aqueous buffer (40 mM MOPS, pH 7.0). After dilution,
aliquots were withdrawn at various times for assay of ATP and
PP
. In B, after F
(2.3 µM) had
been allowed to form the indicated amounts of ATP and PP
for 4 h (time zero), the mixture was diluted with 6 volumes of
aqueous buffer, and an aliquot was placed on Centricon filters and
centrifuged for 45 min. At this time, ATP and PP
were
determined in the whole diluted mixture and in the filtrate (ND, not
detectable). The results are expressed as nmol
[
P]P
incorporated into ATP or
PP
per ml of original synthetic mixture.
Table: Effect of dilution on ATP and PP synthesized by F
in complex with its
inhibitor protein
in complex with its inhibitor
protein (3.2 µM) was incubated in the standard synthetic
mixture for 4 h. The amount of ATP and PP
formed were
determined. At this time, the mixture was diluted with aqueous buffer,
and an aliquot was centrifuged over Centricon filters (1 h). The amount
of ATP and PP
were determined in both the diluted mixture
that had not been centrifuged and in the filtrate. After 4 h, the
synthetic mixture was also diluted with aqueous buffer that contained
inorganic pyrophosphatase; after 5 min, the reaction was quenched with
trichloroacetic acid, and the amount of ATP and PP
was
determined. ND, not detected. The results are expressed per ml of
original synthetic mixture.
SO, dimethyl sulfoxide;
F
-IP, soluble F
in complex with its inhibitor
protein; MOPS, 3-(N-morpholino)propanesulfonic acid.
P]P
incorporated into ATP, and
PP
disappeared. This is because hexokinase (Sigma type IV)
at the concentration used exhibits substantial pyrophosphatase activity
(Tuena de Gómez-Puyou et al., 1993).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.