From the
Binding of pyrophosphate (PP
P
Escherichia coli F
Purified E. coli F
A parallel line
of reasoning and experimentation allowed us to characterize the
noncatalytic sites. Residue Arg-365 of the
Pyrophosphate (PP
The availability of mutant E. coli enzymes with reporter tryptophans substituted specifically at the
catalytic or the noncatalytic sites provides a new technique which can
be used to study PP
CB5 F
We carried out
further experiments to investigate this phenomenon. CB5 F
In contrast, when
NaPP
These experiments indicated
that displacement of nucleotides from F
The experiments were
repeated using a preincubation period of 1 h instead of 20 s. No
difference was seen with the enzyme preloaded with MgGTP
(k
Native AW7 F
Free PP
Our finding that the K
Binding parameters for
MgAMP-PNP were obtained by fitting theoretical curves to the data shown
in Fig. 2. A good fit was obtained assuming two types of binding sites
with N
We thank Cheryl Bowman for excellent technical
assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) to the three catalytic
(``C'') and three noncatalytic (``NC'') nucleotide
sites of Escherichia coli F
-ATPase was determined
by fluorescence spectroscopy using mutant enzymes with tryptophan
inserted specifically in either C sites (
Y331W) or NC sites
(
R365W). Fluorescence of the tryptophan is quenched on binding of
nucleotide; PP
binding parameters were determined by
competition with ATP or adenyl-5`-yl imidodiphosphate. It was found
that MgPP
binds to each NC site with
K
= 20 µM. In
contrast, even at millimolar concentration, neither MgPP
nor free PP
showed significant binding to C sites. We
confirmed that free PP
displaces nucleotide from C sites,
but this was shown to be due to complexation of Mg
ions rather than to occupancy of the sites. MgPP
bound at NC sites was found not to affect ATP hydrolysis rates.
From the data we propose a two-phase model for nucleotide binding at NC
sites. In phase one, NC sites recognize the pyrophosphate
``end'' of the nucleotide, which binds initially with
K
similar to MgPP
; in phase
two, a slow conformational change occurs which tightly sequesters
adenine nucleotide. Phase two does not occur with guanine nucleotide.
This model explains the preference of NC sites for adenine nucleotides.
(5 mM) did not bind to either C or NC sites.
F
-ATP
synthase is the enzyme responsible for ATP synthesis in oxidative
phosphorylation and generation of a transmembrane proton gradient by
ATP hydrolysis. It consists of a membrane-embedded F
sector
attached by a stalk to the F
sector, which projects from
the membrane on the cytoplasmic face of the bacterial plasma membrane
(for recent reviews, see Senior, 1990; Fillingame, 1990; Capaldi et
al., 1994). The F
sector may readily be released from
membranes in low ionic strength buffer and purified to homogeneity as
an active ATPase (e.g. Senior et al., 1979).
-ATPase contains a total of
six nucleotide-binding sites, of which three are catalytic, and three
are relatively non-exchangeable and noncatalytic (Wise et al.,
1983; Perlin et al., 1984; Issartel et al., 1986). As
reviewed in Weber et al. (1993a), earlier affinity and
photoaffinity labeling experiments and mutagenesis studies in several
laboratories had suggested that residue Tyr-331
(
)
of the F
-subunit was located in the
catalytic site, in close proximity to bound ATP. Use of the fluorescent
substrate analogs lin-benzo-ADP and lin-benzo-ATP in
our laboratory indicated that residue
-Tyr-331 makes direct
contact with the base moiety of bound analog (Weber et al.,
1992). Substitution of tryptophan, in the
Y331W mutant, generated
an F
enzyme which retained catalytic activity similar to
that of wild-type, and the fluorescence of the 3
-Trp-331 residues
(in the three
-subunits) provided a specific and direct probe of
nucleotide binding to catalytic sites (Weber et al., 1993a).
By this technique it was shown that all three catalytic sites must be
occupied to achieve physiological (V
) rates of
ATP hydrolysis and that occupation of two sites leads, at most, to a
very slow rate of ATP hydrolysis. Also, binding parameters for MgATP,
MgADP, and MgAMP-PNP
(
)
at each of the three
catalytic sites were reported; with all three nucleotides, the
catalytic sites showed negative binding cooperativity. In further work
(Weber et al., 1994a) the Mg dependence and nucleotide
specificity of the catalytic sites and effects of inhibitors and
mutations were established. X-ray crystallography studies of the bovine
mitochondrial F
-ATPase enzyme have recently defined the
structure and environment of the three catalytic sites in detail and
have confirmed that residue
-Tyr-331 (actually
-Tyr-345 in
bovine) does indeed lie immediately adjacent to the purine ring of
bound nucleotide (Abrahams et al., 1994).
-subunit appeared from
sequence comparison to be homologous to residue
-Tyr-331, and by
mutagenesis and photoaffinity-labeling studies we confirmed the
location of residue
-Arg-365 as part of the noncatalytic site
(Weber et al., 1993b). F
obtained from the
R365W mutant was found to retain catalytic activity similar to
wild-type, and in this enzyme the newly inserted tryptophan provided a
direct and specific fluorescent probe of nucleotide binding at
noncatalytic sites (Weber et al., 1994b). Using this signal we
established the Mg dependence, nucleotide specificity, and
nucleotide-binding parameters at noncatalytic sites and demonstrated
that occupancy of noncatalytic sites is not required for ATP
hydrolysis. X-ray crystallography studies of the bovine mitochondrial
F
-ATPase enzyme have since defined the structure and
environment of the noncatalytic sites in detail and have confirmed that
residue
-Arg-365 (actually
-Arg-362 in bovine) lies
immediately adjacent to the adenine ring of bound nucleotide (Abrahams
et al., 1994).
) is
potentially a valuable probe of functional characteristics of
F
-ATPase nucleotide-binding sites in wild-type and mutant
enzymes. Studies with bovine mitochondrial enzyme have demonstrated
that up to 3 mol of [
P]PP
may become
bound per mol F
(Kironde and Cross, 1986; Issartel et
al., 1987; Peinnequin et al., 1992; Jault and Allison,
1993). In the paper of Kironde and Cross(1986), PP
was
shown to promote release of adenine nucleotide from catalytic sites,
and it was concluded that PP
bound to two catalytic sites (
of Kironde and Cross(1986)). However, later work from the
same laboratory (Milgrom and Cross, 1993) has indicated that PP
became bound to noncatalytic sites and appeared not to compete
effectively with GTP, ATP, or ADP for binding to catalytic sites. Jault
and Allison(1993) also made the interpretation that PP
binds at noncatalytic sites, whereas Peinnequin et al. (1992) concluded that PP
binds to specific sites which
interact with and influence occupancy of catalytic and noncatalytic
sites, but which are distinct from both. Therefore, the current
literature is conflicting as to the location of PP
-binding
sites. In bovine mitochondrial, F
binding constants
(K
) for PP
of approximately
1-20 µM (Issartel et al., 1987; Peinnequin
et al., 1992), or 15-230 µM (Kironde and
Cross, 1986) have been reported. Studies of interaction of PP
with E. coli F
have been fewer. A recent
paper (Hyndman et al., 1994) showed that PP
competed effectively for binding of GTP or ATP to noncatalytic
sites, but had little or no effect on binding to catalytic sites.
PP
binding parameters have not been reported for E.
coli F
.
binding. In this work, we have used the
technique to study the sites at which PP
competes with
nucleotide for binding and to determine K
values for PP
. The data demonstrate a major
difference between catalytic and noncatalytic sites and suggest that,
in the binding process, the noncatalytic sites recognize initially the
pyrophosphate ``end'' of nucleotide ligands. We offer also an
explanation for the effect of PP
to promote net loss of
nucleotide from catalytic sites, based on its ability to complex
Mg
ions rather than by direct competition for
occupancy of the sites.
Fluorescence Measurements
These were done as
described in Weber et al. (1993a, 1994a, 1994b). The buffers
used in fluorescence experiments are described in the figure legends
and in the text. Before use F enzymes were pre-equilibrated
in buffer in one of two ways as follows. 1) Mg
-free
buffer: the enzyme was passed sequentially through two 1-ml Sephadex
G-50 centrifuge columns in 50 mM Tris-SO
, pH 8.0,
at 23 °C. 2) Mg
-containing buffer: the enzyme was
passed through one 1-ml Sephadex G-50 column in 50 mM
Tris-SO
, pH 8.0, 2.5 mM MgSO
, at 23
°C.
F
These were done as described in Weber et
al. (1994b). The procedure for preparation of nucleotide-depleted
enzyme was as in Senior et al.(1992).
Purification and Characterization: Assays
of Enzyme Function
F
Enzyme from strain SWM4
( Enzymes
Y331W mutant) was as in Weber et al. (1993a); from
strain AW7 (
R365W mutant) as in Weber et al. (1994b);
from strain SWM1 (wild-type) as in Rao et al. (1988a). Strain
CB5 was constructed as follows. Plasmid pOW1 (Wilke-Mounts et
al., 1994) was digested with SstI and the larger (vector)
fragment was purified. Plasmid pSWM4 (Weber et al., 1993a) was
digested with SstI, and the smaller fragment containing the
Y331W mutation was purified. The two purified fragments were
ligated together and transformed into strain TG1rA (Rao et
al., 1988b). Plasmid minipreps obtained from transformants were
screened first with PstI to determine correct orientation of
the SstI-SstI insert, and then with NaeI,
which identifies the
Y331W mutation. One plasmid, named pCB5, was
subjected to DNA sequencing to confirm that the
Y331W mutation was
present, and then transformed into strain AN888 (Downie et
al., 1981), yielding strain CB5. Plasmid pCB5 contains six total
mutations. In addition to the
Y331W mutation at the catalytic
site, it contains the mutations
W28L/
W513F/
W108Y/
W206Y/
W107F which together
remove all the tryptophans naturally present in wild-type
F
.
F
SWM4 F Enzymes Used in This Work
(
Y331W mutant) contains the 9
wild-type tryptophans plus 3 additional tryptophans at residue
-331. The properties of this enzyme are similar to wild-type and
have been described by Weber et al. (1993a, 1994a). The
-Trp-331 fluorescence signal reports occupancy of the catalytic
sites by nucleotide. AW7 F
(
R365W mutant) contains the
9 wild-type tryptophans plus 3 additional tryptophans at residue
-365. The properties of this enzyme are also similar to wild-type
and have been described by Weber et al. (1994b). The
-Trp-365 fluorescence signal reports occupancy of the noncatalytic
sites by nucleotide.
contains none of the
wild-type tryptophans, it contains only 3 tryptophans at position
-331 (see ``Materials and Methods'' for construction of
strain CB5). The entire fluorescence signal (
= 295 nm) in this enzyme is due to
-Trp-331 at the
catalytic sites. Strain CB5 showed the same growth characteristics as
wild-type on succinate plates and in liquid medium containing limiting
(3 mM) glucose. CB5 F
was found to show normal
molecular size by Sephacryl S-300 chromatography and normal subunit
composition on SDS-gels. V
for ATP hydrolysis
was 70% of wild-type value, and
k
/K
(1.8
10
M
s
) was
similar to that of wild-type (1.9
10
M
s
).
Studies of Interactions of PP
with Catalytic
Sites
Binding of Free ATP to Catalytic Sites in Presence and
Absence of 1 mM Free PP
In these
experiments, SWM4 F (
Y331W) was used. The enzyme was
pre-equilibrated with Mg
-free buffer by passage
through two centrifuge columns (see ``Materials and
Methods''). The residual adenine nucleotide occupancy at catalytic
sites was
0.2 mol/mol, as indicated by the fluorescence signal.
Fluorescence spectra were recorded in EDTA-containing buffer, and
different concentrations of ATP were added to elicit varying degrees of
catalytic site occupancy, as indicated by quenching of the fluorescence
signal. The fluorescence responses to ATP were similar to those
reported previously (Weber et al., 1994a), and it may be noted
that it was also shown previously that ATP hydrolysis activity was
essentially zero under these conditions. The presence of 1 mM
NaPP
had no effect on the ATP-induced fluorescence
responses (Fig. 1) indicating that free PP
did not
bind to catalytic sites.
Figure 1:
Effect of free
PP on binding of free ATP to the catalytic sites of E. coli
F
. SWM4 F
(
Y331W) was titrated with ATP in
a buffer containing 50 mM Tris-SO
, 0.5 mM EDTA, pH 8.0, either in absence (
) or in presence (
)
of 1 mM NaPP
. ATP binding stoichiometries were
calculated from the decrease in fluorescence of residue
-Trp-331.
The solid line represents a fit to the data points obtained in
absence of NaPP
(2.9 equivalent binding sites with K = 71 µM).
Binding of MgAMP-PNP to Catalytic Sites in Presence and
Absence of 1 mM MgPP
In these experiments,
SWM4 F (
Y331W) was titrated with AMP-PNP in a buffer
containing 2.5 mM Mg
. The enzyme was
pre-equilibrated with Mg-free buffer by passage through two centrifuge
columns, such that the residual adenine nucleotide occupancy at
catalytic sites was
0.2 mol/mol, as indicated by the fluorescence
signal. The binding curve for MgAMP-PNP seen in absence of MgPP
(Fig. 2) was the same as that reported previously in Weber
et al. (1993a). The presence of 1 mM MgPP
had very little, if any, effect on the MgAMP-PNP binding curve
(Fig. 2). summarizes the binding parameters obtained
from the Fig. 2data. There was no significant MgPP
binding to the two catalytic sites of lower affinity. In the case
of the high-affinity site, we cannot exclude the possibility of very
weak MgPP
binding; however, the dissociation constant of
this site for MgPP
is at least 1 mM. Attempts to
obtain more precise data by increasing the MgPP
concentration failed due to formation of precipitate in the
cuvette.
Figure 2:
Effect of MgPP on binding of
MgAMP-PNP to the catalytic sites. SWM4 F
(
Y331W) was
titrated with AMP-PNP in a buffer containing 50 mM
Tris-SO
, 2.5 mM MgSO
, pH 8.0, either
in absence (
) or in presence (
) of 1 mM MgPP
(1 mM NaPP
plus 1 mM
MgSO
, increasing the total MgSO
concentration
to 3.5 mM). MgAMP-PNP binding stoichiometries were calculated
from the decrease in fluorescence of residue
-Trp-331. The
solid line represents a fit to the data points obtained in
absence of PP
, the dashed line to those obtained
in its presence. The resulting binding parameters are given in Table
I.
Apparent Displacement of Catalytic Site-bound Nucleotide
by PP
When SWM4 F was pre-equilibrated
by passage through a single centrifuge column in buffer containing
Mg
ions (2.5 mM) the residual adenine
nucleotide occupancy at catalytic sites was 0.4-0.8 mol/mol
F
, as indicated by the fluorescence signal. We previously
reported (Weber et al., 1993a) that addition of 3 mM
NaPP
to this enzyme, in a buffer containing 2.5 mM
Mg
ions, caused an increase in fluorescence (by
5-10%), indicating that PP
caused displacement of
adenine nucleotide from catalytic sites. This confirmed the work of
Kironde and Cross(1986), who showed that, in bovine mitochondrial
F
, 5 mM NaPP
displaced adenine
nucleotides from catalytic sites when added to enzyme in a buffer
containing 2 mM Mg
ions. Given that neither
free PP
nor MgPP
competes for binding of
nucleotide to catalytic sites (above), it appeared that the
displacement of nucleotides from catalytic sites was not due to actual
occupancy of catalytic sites by PP
.
was used in this part of the work because in this enzyme the
entire fluorescence signal (
= 295 nm) is
attributable to the substituted
-Trp-331; all of the wild-type
tryptophans have been removed by mutagenesis. This eliminates any
possibility that fluorescence changes engendered by PP
are
due to effects on tryptophans other than at residue
-331. CB5
F
was first pre-equilibrated by passage through two
centrifuge columns in Mg-free buffer, after which it was found to
contain
0.2 mol/mol adenine nucleotide at catalytic sites as
indicated by the fluorescence signal. Fig. 3shows the
fluorescence spectrum of this enzyme in absence of added nucleotide
(curve 1). It may be remarked that this spectrum is very
similar to the difference spectrum seen when comparing SWM4 F
to wild-type enzyme (as in Fig. 2B of Weber et
al. (1993a)), confirming that the fluorescence spectrum of SWM4
F
is indeed the sum of the independent contributions of the
residue
-331 tryptophans and those present in wild-type
F
; this means that no significant fluorescence energy
transfer occurs between both sets of the fluorophore. Addition of 1
mM MgATP to CB5 F
(Fig. 3, curve 2)
caused almost complete quenching of the fluorescence
signal.
(
)
Addition of 1 or 10 µM
MgATP caused partial quenching of fluorescence (curves 3 and
5) indicating catalytic sites occupancy of 1.2 and 1.9
mol/mol, respectively. The presence of 1 mM MgPP
did not change the response to 1 µM MgATP (curve
4) or 10 µM MgATP (curve 6), demonstrating
that MgPP
did not compete with MgATP for binding at
catalytic sites.
Figure 3:
Effect of
MgPP on ATP-induced quenching of CB5 F
fluorescence. Shown are uncorrected emission spectra (
= 295 nm) of CB5 F
(50 nM in 50
mM Tris-SO
, 2.5 mM MgSO
, pH
8.0). Curve 1 without added nucleotide (±1 mM
MgPP
); curve 2 with 1 mM ATP; curves
3 and 4 with 1 µM ATP; curve 5 and
6 with 10 µM ATP. 1 mM MgPP
was present in curves 4 and
6.
CB5 F was then prepared by
pre-equilibration in buffer containing 2.5 mM Mg
ions by passage through one centrifuge column. This preparation
retained about 0.8 mol/mol adenine nucleotide at catalytic sites as
judged by the fluorescence signal. Addition of NaPP
(3
mM final concentration) to this enzyme in buffer containing
2.5 mM Mg
ions increased the fluorescence
signal by 11-15%, indicating that release of nucleotide from
catalytic sites had occurred. A 20-23% increase in fluorescence
was obtained by addition of EDTA, instead of NaPP
, to a
final concentration of 3 mM and addition of EDTA to a final
concentration of 6 mM increased the fluorescence by a total of
30%. The final level of the fluorescence signal in the latter case
indicated complete emptying of catalytic sites.
and MgSO
(both 3 mM) were added
together, the fluorescence increase seen was smaller (6-8%) than
with NaPP
alone. The same final level of fluorescence was
reached if the MgSO
was added after the NaPP
.
Attempts to achieve a higher excess of Mg
concentration over PP
were confounded by
precipitation in the cuvette, which caused an artifactual increase in
the fluorescence signal. When EDTA and MgS0
(both 3
mM) were added together, the fluorescence increase seen was
only 6% as contrasted to 20-23% with EDTA alone, and if 10
mM MgS0
was added with 3 mM EDTA, there
was no increase in fluorescence at all.
catalytic sites by
PP
is related not to actual binding of PP
at
the sites but rather to its ability to complex Mg
ions. This is reasonable, given that the nucleotide species bound
at catalytic sites in native enzyme after purification is ADP (Kironde
and Cross, 1986; Senior et al., 1992), that the stability
constants at pH 8.0 for MgADP and MgPP
complexes are
similar (pK(MgADP) = 3.6, pK(MgPP
)
= 3.7; see O'Sullivan and Smithers, 1979; Dawson et
al., 1984), and that the affinity of catalytic sites for adenine
nucleotide is strongly enhanced in presence of Mg
(Weber et al., 1994a). Since the effect of PP
may be readily mimicked by EDTA, and the enzyme prepared under
the conditions described above contains noncatalytic sites essentially
fully occupied by endogenous nucleotide (Weber et al., 1994b),
this effect of PP
does not involve the noncatalytic sites.
Studies of Interactions of PP
with
Noncatalytic Sites
Binding of MgAMP-PNP to Noncatalytic Sites in Presence
and Absence of PP
Since binding of
nucleotides to noncatalytic sites is Mg-dependent (Weber et
al., 1994b), we did not conduct any studies in absence of
Mg or P
ions. Nucleotide-depleted AW7 enzyme (
R365W
mutant) was prepared, pre-equilibrated with buffer containing 2.5
mM Mg
, and titrated with AMP-PNP either in
the absence of, or in the presence of, 5 mM P
, 100
µM PP
or 200 µM PP
.
Fig. 4
shows the data obtained. The data were fitted according to
a binding model assuming N equivalent and independent binding
sites for MgAMP-PNP. A good fit was obtained, in absence of P
or PP
, with N = 2.8 and
K
= 16 µM.
Previously, we found that the same binding model (3.0 and 2.8
equivalent sites, respectively) described well the binding of MgATP
(K
= 25 µM) and MgADP
(K
= 24 µM) to
noncatalytic sites of AW7 F
(Weber et al., 1994b).
It is seen that 5 mM P
had zero effect on
MgAMP-PNP binding (Fig. 4, open squares), whereas 100
µM PP
(closed circles) and 200
µM PP
(triangles) competed for
binding at all three sites. The apparent
K
(MgPP
) calculated from the
data in Fig. 4was 20 µM at each site (average of
all data). Thus, it was evident that the
K
(MgPP
) was similar to that
for Mg nucleotides.
Figure 4:
Effect of P and
MgPP
on MgAMP-PNP binding to the noncatalytic sites.
Nucleotide-depleted AW7 F
(
R365W) was titrated with
AMP-PNP in a buffer containing 50 mM Tris-SO
, 2.5
mM MgSO
, pH 8.0; (
) in absence of P
or PP
; (
) with 5 mM P
,
(
) with 100 µM PP
, (
) with 200
µM PP
. MgAMP-PNP binding stoichiometries were
calculated from the decrease in fluorescence of residue
-Trp-365.
The solid line represents a fit to the data points obtained in
absence of P
or PP
(2.8 identical sites with
K = 16 µM), the dotted and
dashed lines are fits to the data points for 100 and 200
µM PP
,
respectively.
Therefore several points have been established.
First, MgAMP-PNP binds to all three F noncatalytic sites,
in agreement with previous work which utilized radioactive MgAMP-PNP
(Wise et al., 1983), and similar to MgATP and MgADP (Weber
et al., 1994b). Second, the presence of 5 mM P
had zero effect on MgAMP-PNP binding, showing that P
does not bind at noncatalytic sites. Third, it is evident that
MgPP
does compete for binding of MgAMP-PNP at all three
noncatalytic sites. Fourth, the apparent binding affinity for
MgPP
at noncatalytic sites is similar to that for Mg
nucleotides.
Use of a MgPP
Nucleotide-depleted
AW7 F Trap to Measure Dissociation of
Bound Nucleotides from Noncatalytic Sites
was incubated in buffer containing 50 mM
Tris-SO
, 2.5 mM MgSO
, pH 8.0, for 20 s
in presence of 1 mM ATP or GTP. At this concentration of
nucleotide, >2 noncatalytic sites were filled in the MgATP-reloaded
enzyme and 1.1 in MgGTP-reloaded F
(consistent with
previous data of Weber et al., 1994b). The enzyme solutions
were then diluted 200-fold into the same buffer containing 0.2
mM NaPP
, such that final enzyme concentration was
50 nM, and the fluorescence signals were monitored. With
MgGTP-reloaded F
, the fluorescence signal reached
immediately the same level as that of the control (preincubated without
nucleotides). This means that virtually all GTP had been released
during mixing time (about 15 s). Thus, we could only estimate an upper
limit of 5 s for the apparent half-time for dissociation of GTP from
the noncatalytic sites (k
>0.1
s
). In contrast, release of ATP was much slower. The
apparent half-time for dissociation of ATP was 8-11 min
(k
= 1.0-1.4
10
s
).
>0.1 s
), but with MgATP
the k
decreased to 2-4
10
s
. (Since 5 µM
nucleotide was present in the medium during the dissociation phase of
the experiment, these values are apparent values only, however, it
should be noted that when ATP-loaded F
was passed through a
centrifuge column immediately before the dilution step, the same
results were obtained).
, in which the
noncatalytic sites were already nearly fully occupied by endogenous
adenine nucleotide (2.7 mol/mol F
), was incubated with 1.0
mM NaPP
in 50 mM Tris-SO
, 2.5
mM MgSO
, pH 8.0, 23 °C, and it was seen that
over a period of 3 h there was no measureable release of nucleotide
from the noncatalytic sites. Therefore in the native AW7 enzyme the
dissociation rate of adenine nucleotide is very slow indeed.
Additionally, inclusion of 50 µM ATP to induce enzyme
turnover did not cause nucleotide release.
Effects of MgPP
Using wild-type F on ATP
Hydrolysis
we tested the effects
of MgPP
in assays of steady-state ATP hydrolysis. In these
experiments, nucleotide-depleted enzyme was compared with native
F
; the latter preparation contained noncatalytic sites
essentially fully loaded with adenine nucleotides (Weber et
al., 1994b). Both enzymes were preincubated with 1 mM
NaPP
in buffer containing 50 mM
Tris-SO
, 2.5 mM MgSO
, pH 8.0, 23
°C, then diluted into ATPase assay medium (final concentrations: 50
mM Tris-SO
, pH 8.0, 1 mM PP
,
10-2000 µM ATP, MgSO
equal to the sum of
PP
and ATP concentrations). There was no significant
difference in ATPase activity between the native and
nucleotide-depleted enzymes. Under the conditions of the experiment,
the noncatalytic sites of the nucleotide-depleted enzyme would have
been occupied by MgPP
, and we may conclude that this did
not affect the rates of catalysis to a significant extent.
General Comments
The use of mutant E. coli F-ATPase enzymes containing tryptophans substituted
specifically in catalytic sites (
Y331W) or noncatalytic sites
(
R365W) has enabled us to determine at which of these sites
PP
or P
compete with nucleoside triphosphate
for binding, and to determine apparent K
values. summarizes the data for binding of
different ligands to the catalytic and noncatalytic sites, derived from
work presented here and from previous reports (Weber et al.,
1993a, 1994a, 1994b). The data for MgPP
are shown in
, line 5. It is seen that MgPP
binds at
noncatalytic sites, and the apparent
K
(MgPP
) of 20 µM
is surprisingly similar to that for MgAMP-PNP, MgATP, and MgADP, as
measured by the tryptophan fluorescence signal. Occupancy of the
noncatalytic sites by MgPP
had no effect on ATP hydrolysis.
Neither MgPP
() nor free PP
(see
``Results'') was seen to bind significantly to catalytic
sites. P
(in presence of Mg) did not bind to either type of
site (). Further to the data in , it may be
noted that MgGTP binds to both types of site whereas MgGDP binds to
catalytic but not to noncatalytic sites (Weber et al., 1994a,
1994b; Hyndman et al., 1994)
caused net
displacement of nucleotide from catalytic sites of native enzyme; this
effect was mimicked by EDTA and is apparently related to the ability of
PP
to complex Mg
ions, as discussed
below. There was no measurable release of nucleotide from noncatalytic
sites of native enzyme in presence of PP
; however release
of nucleotide from noncatalytic sites of enzyme which had been
nucleotide-depleted then reloaded was faster. MgPP
was used
as a trap to allow determination of relative dissociation rates for
reloaded MgGTP and MgATP. Based on the results, a model for nucleotide
binding to noncatalytic sites is described below.
The Binding Process at Noncatalytic
Sites
Nucleotide binding at noncatalytic sites shows anomalous
features. For example, when monitored by the -Trp-365 fluorescence
signal, binding of MgATP to AW7 F
occurred with an apparent
K
= 25 µM and
k
= 270 M
s
(Weber et al., 1994b). As was
discussed in that paper, this leads to a calculated k
value of 6.8
10
s
,
which is much faster than expected from previous work in which
nucleotide-depleted wild-type F
was reloaded with
radioactive MgATP and dissociation of radioactivity followed (Pagan and
Senior, 1990), and also much faster than the dissociation of
nucleotides from the noncatalytic sites of native AW7 F
(this paper). We suggested in Weber et al., (1994b)
that, for MgATP, subsequent to the initial binding event which is
recorded by the fluorescence signal, a conformational change occurs in
the noncatalytic site which has the effect of considerably reducing
k
. It was further proposed that guanine
nucleotide is not able to bring about this conformational change, and
so the apparent k
for MgGTP remains relatively
high.
for
MgPP
is similar to that for MgATP, MgADP and MgAMP-PNP,
when measured by the fluorescence technique () leads us to
propose that in the initial phase of binding of nucleotide at
noncatalytic sites it is the pyrophosphate ``end'' of the
nucleotide that is recognized. The initial phase of binding is proposed
to lead to ``State I'' in which the site is now occupied, but
the nucleotide has a relatively fast k
. Then, if
the nucleotide contains adenine as the base, a conformational change is
induced which sequesters the nucleotide in ``State II,''
rendering it ``non-exchangeable'' by reducing
k
. This conformational change does not
apparently occur with guanine nucleotide. Supportive of this, using
MgPP
as a trap to prevent rebinding of nucleotide, we have
shown here that in enzyme that had been depleted of endogenous
nucleotides and then reloaded, the dissociation rate of MgGTP is much
faster than that of MgATP. Also, the dissociation rate measured for
MgATP reloaded into nucleotide-depleted F
was lower than
that calculated from the K
(MgATP)
(above). Prolonging the MgATP ``reloading period'' decreased
the dissociation rate further. Still, however, it was considerably
higher than that for dissociation of endogenous nucleotides (usually a
mixture of ADP and ATP; see Wise et al., 1981; Issartel et
al., 1986; Senior et al., 1992) from native
F
. Thus it appears as if there are additional factors
involved in the native enzyme that decrease nucleotide dissociation
rates from noncatalytic sites.
Functional Comparisons between Catalytic and Noncatalytic
Sites
In both catalytic and noncatalytic nucleotide-binding
sites of F, residues from the ``Homology A'' and
``Homology B'' consensus sequences play a prominent role in
binding the phosphate moieties of the nucleotides (Abrahams et al. 1994). One difference between the structures in the two types of
site that might explain the presence of catalytic activity in the
former but not in the latter has been discussed by Abrahams et
al., who suggested that residue
-Glu-181 of the catalytic
site may be critical. This suggestion is fully consistent with earlier
kinetic and thermodynamic analyses of catalytic properties of the
E181Q mutant enzyme (Senior and Al-Shawi, 1992). The equivalent
residue in the noncatalytic site is
-Gln-208. A second difference
between the two types of site is that the noncatalytic sites are
relatively specific for adenine nucleotide (Perlin et al.,
1984; Weber et al., 1994b) in comparison to the catalytic
sites. Abrahams et al.(1994) suggest that this could be due to
hydrogen-bonding properties of the residues surrounding the base moiety
of the nucleotide in the two types of site. The dissociation rates for
MgGTP versus MgATP (above) provide experimental explanation
for this apparent specificity of the noncatalytic sites. A third
difference is that the catalytic sites bind nucleotides not only in the
presence but also in the absence of Mg
, albeit with
altered characteristics; in contrast, the noncatalytic sites are
strongly Mg
-dependent (Weber et al., 1994a,
1994b). The work presented here defines a fourth, clear-cut, difference
between the catalytic site and the noncatalytic site, namely that
significant binding of MgPP
is seen only in the latter. A
structural rationale for this difference is not yet known, and its
elucidation will be of considerable interest. From this work it is
apparent that measurements of MgPP
binding parameters in
wild-type and particularly mutant E. coli enzymes can now be
used as a distinguishing functional characteristic of the noncatalytic
sites. We anticipate that this will facilitate studies of the
physiological role of the noncatalytic sites which, despite much
research, remains obscure.
Displacement of Nucleotides from the Catalytic Sites by
PP
We confirmed here previous results showing that
PP Is Due to Complexation of Mg
Ions
was apparently able to displace nucleotide from the
catalytic sites, and yet we found that neither free PP
nor
MgPP
was able to bind to catalytic sites. The explanation
for this conundrum appears to reside in the fact that PP
complexes with Mg
ions relatively strongly. In
a solution of native F
, MgADP will be continually released
from the catalytic sites of the enzyme into the medium. Whereas this
nucleotide could normally rebind readily as MgADP, in the presence of
an excess of PP
some of the MgADP will lose Mg
to PP
, and consequently its rebinding will be
impaired. Thus the apparently conflicting data in the literature are
satisfactorily resolved.
Filling of the Noncatalytic Sites with MgPP
In this study we demonstrate
that MgPP Has No Effect on Catalysis
binding at noncatalytic sites had no effect on
ATP hydrolysis in E. coli F
. Jault and Allison
(1993) have suggested that the effect of PP
to increase the
ATPase activity in bovine mitochondrial F
-ATPase is caused
by the fact that binding of PP
into the three noncatalytic
sites induces release of long-lived inhibitory MgADP from a catalytic
site. In a previous study we demonstrated that long-lived MgADP
inhibition is not found in E. coli F
as prepared
here (Senior et al., 1992). Therefore the lack of an effect of
PP
in E. coli F
is not unexpected.
Given that MgPP
did not displace adenine nucleotide from
the noncatalytic sites of native F
(above) and taking into
account the ambient cytoplasmic ATP concentrations of 2-3
mM in E. coli cells, it is not likely that MgPP
plays a role in physiological regulation of
F
F
. The major value of this study therefore is
to show that PP
can be a valuable experimental tool for
stucture-function studies of the nucleotide sites.
Table:
Binding of MgAMP-PNP to the catalytic sites in
absence and presence of MgPP
= 1.0 and N
= 1.5, both in absence and presence of MgPP
.
Dissociation constants are given ± standard deviation.
Table:
Binding of ligands to
nucleotide sites in E. coli F as determined by fluorescence
signal of
W365 or
W331 residues
preparation.
Quantification of the total tryptophan content after denaturation in 6
M guanidine hydrochloride gave a value of 4.0 mol/mol
F
, rather than 3 mol/mol as expected. This means that the
protein contamination would amount to approximately 2% if an average
tryptophan content is assumed (see Wilke-Mounts et al. (1994)).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.