(Received for publication, May 15, 1995; and in revised form, June 20, 1995)
From the
X-ray structure analysis of the noncatalytic sites of
F-ATPase revealed that residue
-Asp
lies
close to the Mg of bound Mg-5`-adenylyl-
,
-imidodiphosphate.
Here, the mutation
D261N was generated in Escherichia coli and combined with the
R365W mutation, allowing nucleotide
binding at F
noncatalytic sites to be specifically
monitored by tryptophan fluorescence spectroscopy. Purified
D261N/
R365W F
-ATPase showed catalytic activity
similar to wild-type. An important feature was that, without any resort
to nucleotide-depletion procedures, the noncatalytic sites in purified
native enzyme were already empty. Binding studies with MgATP, MgADP,
and the corresponding free nucleotides led to the following
conclusions. Residue
-Asp
interacts with the Mg of
Mg-nucleotide in noncatalytic sites and provides a large component of
the binding energy (
3 kcal/mol). It is the primary determinant of
the preference of noncatalytic sites for Mg-nucleotide. The natural
ligands at these sites in wild-type enzyme are the Mg-nucleotides and
free nucleotides bind poorly.
Under conditions where noncatalytic
sites were empty, D261N/
R365W F
showed
significant hydrolysis of MgATP. This establishes unequivocally that
occupancy of noncatalytic sites by nucleotide is not required for
catalysis.
FF
-ATP synthase is the enzyme
responsible for ATP synthesis by oxidative phosphorylation and
ATP-driven proton extrusion in Escherichia coli. The catalytic
F
-sector is an oligomer with subunit composition
, which contains six
nucleotide-binding sites (Wise et al., 1983). Three sites are
catalytic sites, whose structure is provided primarily from the
-subunits as shown by the x-ray structure resolution of bovine
F
(Abrahams et al., 1994). Simultaneous occupancy
of all three catalytic sites is necessary for physiological rates of
ATP hydrolysis (Weber et al., 1993a). The other three sites
are noncatalytic sites, formed primarily from
-subunits (Abrahams et al., 1994). Although the ligand-binding properties of these
sites have been established in some detail (e.g. see Perlin et al.(1984), Milgrom and Cross (1993), Jault and
Allison(1993), Weber et al.(1994), and Weber and
Senior(1995)), their physiological role has proved difficult to
ascertain.
One obstacle in studying the noncatalytic sites has been
of a technical nature. Although it was possible to prepare
F-ATPase which was completely depleted of nucleotides, and
therefore had empty noncatalytic sites (Garrett and Penefsky, 1975;
Wise et al., 1983; Senior et al., 1992), there was
until recently no method of specifically monitoring noncatalytic site
occupancy. The use of ``chases'' with GTP or PPi, or with
adenine nucleotides for various times, in order to replace catalytic
site nucleotides and thus allow estimation of noncatalytic site
nucleotides (presumed to be nonexchangeable) is complicated by the
findings that both GTP and PPi bind to noncatalytic sites (Weber et
al., 1994; Hyndman et al., 1994; Weber and Senior, 1995)
and by the fact that catalytic site-bound nucleotide does not rapidly
exchange with medium nucleotide under all conditions.
However, this
problem was recently solved by use of fluorescence responses from a
tryptophan residue specifically placed at residue -365 by
R365W mutagenesis. (
)Residue
-Arg
interacts directly with the adenine ring of bound nucleotide in
noncatalytic sites (Weber et al., 1993b; Abrahams et
al., 1994) (
)and the fluorescence of residue
-Trp
is fully quenched on binding of nucleotide,
providing a direct and specific probe of noncatalytic site occupancy
(Weber et al., 1994).
Here we have studied mutations at two
positions within the F noncatalytic sites which are
expected to disrupt nucleotide binding, namely
-Lys
and
-Asp
. The former lies in the Walker
Homology A (P-loop) sequence and the latter in the Walker Homology B
sequence (Walker et al., 1982). Residue
-Lys
is seen in the x-ray structure of noncatalytic sites to lie
close to the terminal phosphate of bound Mg-AMPPNP (
)(Abrahams et al., 1994). Previous work (Rao et al., 1988; Yohda et al., 1988) showed that the
mutations
K175I and
K175E abolished or greatly reduced
nucleotide binding to the
-subunit. Residue
-Asp
lies close to the Mg moiety of the Mg-nucleotide (Abrahams et
al., 1994), and since nucleotide binding in noncatalytic sites is
Mg-dependent (Weber et al., 1994) it seemed likely that
removal of the carboxyl group by mutagenesis might have large effects.
Yohda et al.(1988) have shown that the
D261N mutation in Bacillus PS3 reduced MgADP binding affinity in isolated
-subunit.
In each case, the ``disrupting'' mutation
was combined with the R365W mutation in order to allow direct
analysis of nucleotide binding by fluorescence measurement. In this
paper, we describe the properties of the
K175E/
R365W,
K175I/
R365W, and
D261N/
R365W mutants.
Oligonucleotide-directed mutagenesis
(Vandeyar et al., 1988) was used to generate the D261N
mutation in an M13 mp18 template which already contained the
R365W
mutation (ClaI-EcoRI fragment from plasmid pAW7
(Weber et al., 1994) ligated into M13 mp18 between AccI and EcoRI sites). The mutagenic oligonucleotide
used was TG ATC ATT TAC AAT GAC CTT TCG AAA CAG GC, where the
underlined base A introduces the mutation
D261N (GAT
AAT)
and the underlined bases T and G introduced a new BstBI site.
Introduction of the desired mutation was screened by digesting RF phage
with BstBI and MscI (which identifies the
R365W
mutation). The mutations were moved into plasmid pDP34N (Weber et
al., 1993a) on a 1.7-kilobase SacII-SacII
fragment, screening for correct orientation with PstI. The
final plasmid was named pCB6 and the whole DNA sequence between the SacII restriction sites in this plasmid was determined and
shown to be wild-type except for the
D261N/
R365W mutations
and the silent mutations associated with the introduced BstBI
and MscI restriction sites. Plasmid pCB6 was transformed into
strain JP2 and the final strain was designated CB6.
Consistent with previous results, the R365W
single mutant grew well on succinate plates and in limiting glucose
medium, and had significant membrane ATPase activity. As we described
previously, F
can be readily purified from this strain and
it is similar to wild-type F
in properties (Weber et
al., 1994).
The K175E/
R365W mutant did not grow on
succinate plates and had a growth yield in limiting glucose the same as
the Unc
control. Its membrane ATPase activity was not
significant. We were unable to prepare F
from this mutant
because in the final step of purification (S-300 gel filtration) there
was no protein peak at the position corresponding to F
.
With the
K175I/
R365W mutant, growth on succinate plates and
in growth yield tests was strong, however, the membrane-ATPase activity
was low (Table 1). We were not able to purify F
from
this mutant either, because again in the S-300 gel filtration step, no
protein peak corresponding to F
was present. Our earlier
studies of the
K175E and
K175I single mutants (Rao et
al., 1988) showed that the former mutation partially impaired
assembly of F
F
in the cells, whereas the latter
mutation allowed assembly but impaired subunit-subunit interaction,
such that both membrane-bound and purified F
tended to
dissociate. We were previously able to purify F
from the
K175E and
K175I single mutants, but only in low yield. The
present results show that combination of the
K175E and
K175I
mutations with
R365W exacerbated their effects. Jounouchi et
al. (1993) reported that five other mutations at residue
-175
had similar effects, in that they all impaired assembly and/or subunit
stability of F
. Yohda et al.(1988) found that the
K175I mutation in Bacillus PS3 F
impaired
subunit interactions. Therefore, it is clear that mutation of residue
-Lys
interrupts correct assembly of the enzyme and
oligomeric stability of F
.
The D261N/
R365W
mutant grew well on succinate plates, had almost normal growth yield,
and had 25% of the membrane ATPase activity of the isogenic wild-type
strain run alongside (Table 1). The membrane ATPase activity was
76% inhibited by dicyclohexylcarbodiimide (150 µM at pH
8.0, 30 °C, for 60 min), which is similar to wild-type. The
membrane vesicles showed normal ATP-dependent pH gradient formation
when assayed using acridine orange fluorescence quenching as described
by Perlin et al.(1983) (data not shown), consistent with the
fact that the specific ATPase activity of the membranes from the
D261N/
R365W mutant (
0.8 µmol of ATP hydrolyzed per
min/mg of protein) was similar to that of a haploid wild-type strain
(Cox et al., 1978).
Yohda et al.(1988) found
previously that the D261N mutation (in Bacillus PS3) did
not significantly affect subunit assembly when the mutant
-subunit
was reconstituted into an
subcomplex. Compared to the
wild-type subcomplex, the mutation caused nearly 5-fold reduction in V
of ATPase activity of the mutant
subcomplex, and abolished apparent negative
cooperativity that was evident in the wild-type in Lineweaver-Burk
plots. The effect on V
was larger than was seen
here in intact F
, but comparisons are difficult because
both the species and enzyme forms are different. In our ATPase assays,
with the intact F
enzymes from wild-type,
R365W, or
D261N/
R365W, plots of Vversus [S] showed no deviation from simple monophasic
Michaelis-Menten kinetics between 5 and 100% of V
. We did not scrutinize the kinetic behavior at
very low substrate concentrations.
The fluorescence properties of
the purified mutant F were interesting. It may be recalled
that in the
R365W single mutant F
as purified
(``native F
'') the noncatalytic sites are
essentially filled with endogenous adenine nucleotide and the
tryptophan fluorescence spectrum (
= 295 nm)
is the same as for wild-type F
, because fluorescence of the
-Trp
residues is fully quenched (Weber et
al., 1994). When
R365W F
is depleted of
nucleotide by gel filtration in 50% (v/v) glycerol-containing buffer,
the marked fluorescence signal of the
-Trp
residues
is revealed. In contrast, in the case of
D261N/
R365W purified
F
, the fluorescence of the
-Trp
residues
was already fully apparent even in the native F
, without
requiring nucleotide depletion (Fig. 1). This showed that the
D261N mutation had a strong effect, and that in native F
from the
D261N/
R365W mutant, the noncatalytic sites
were empty. Addition of high concentrations of nucleotide quenched the
fluorescence of the
D261N/
R365W mutant F
almost
down to the level of the wild-type enzyme, and it is reasonable to
ascribe the residual difference between the signal of quenched
D261N/
R365W mutant F
and wild-type in Fig. 1to the presence of a small amount of contaminating protein
in the mutant enzyme.
Figure 1:
Fluorescence spectra of mutant F preparations. F
was passed once through a 1-ml
centrifuge column containing 50 mM Tris SO
, pH
8.0, at 23 °C before use. The fluorescence spectra are uncorrected
and were taken at protein concentration of 100 nM in 50
mM Tris-SO
, 0.5 mM EDTA, pH 8.0, at 23
°C. Excitation was at 295 nm. Curve 1,
D261N/
R365W F
in absence of nucleotide; curve
2,
D261N/
R365W F
plus 10 mM ADP; curve 3, wild-type F
±
ADP.
Figure 2:
Binding
of free ATP and MgATP to noncatalytic sites of mutant F preparations. F
was passed through a centrifuge
column as described in the legend to Fig. 1. Titration with free
ATP was in 50 mM Tris-SO
, 0.5 mM EDTA, pH
8.0, at 23 °C. Titration with MgATP was in 50 mM
Tris-SO
, 25 mM MgSO
, pH 8.0, at 23
°C, and ATP was added at increasing concentrations up to 25
mM. Panel A, MgATP binding: open
circles,
R365W F
; closed circles,
D261N/
R365W F
. In both cases the lines are fit to
a model assuming n identical and independent sites. For
D261N/
R365W F
the value of n was set at
3. Panel B, free ATP binding: open circles,
R365W F
. The line is a fit to a model
assuming n identical and independent binding sites; closed
circles,
D261N/
R365W F
. The line is
a fit to a model assuming one site of higher and two sites of lower
affinity.
MgATP was seen to bind
with the same affinity to all three noncatalytic sites of R365W
F
(Fig. 2A; Table 2, first line),
similar to previous results (Weber et al., 1994). In
D261N/
R365W F
, MgATP binding affinity was
drastically reduced (Fig. 2A, Table 2, first
line). In all likelihood the affinity was reduced by the same factor at
all three sites, although as seen in Fig. 2A it was
only possible to fill
2 sites under the experimental conditions.
Free ATP was seen to bind with the same affinity to all three sites in
R365W F
(Fig. 2B, open
circles); however, for
D261N/
R365W F
a
better fit was obtained assuming one site of higher and two sites of
lower affinity (Fig. 2B, closed circles). Nevertheless,
as is clear from inspection of Fig. 2B the two enzymes
were not actually very different in overall behavior toward free ATP.
The calculated K
(free ATP) values are
given in Table 2, second line. It is obvious from Fig. 2that free ATP bound with much weaker affinity than MgATP
in
R365W F
, but in contrast, free ATP bound with
similar or slightly higher affinity than MgATP in
D261N/
R365W
F
.
Figure 3:
Binding of free ADP and MgADP to
noncatalytic sites of mutant F preparations. The procedures
were as described in the legend to Fig. 2. Panel A, MgATP binding: open circles,
R365W
F
; closed circles,
D261N/
R365W
F
. Panel B, free ADP binding: open
circles,
R365W F
; closed circles,
D261N/
R365W F
. In all cases the lines are fit to
a model assuming n identical and independent sites. In panel A,
D261N/
R365W F
, the value of n was set to 3.
We studied nucleotide binding to the noncatalytic sites of E. coli F-ATPase using the fluorescence signal of
the genetically engineered residue
-Trp
as a direct
probe of noncatalytic site nucleotide occupancy (Weber et al.,
1994). Residues
-Lys
and
-Asp
are
both known to lie close to bound nucleotide in noncatalytic sites
(Abrahams et al., 1994). The double mutations
K175E/
R365W,
K175I/
R365W, and
D261N/
R365W
were constructed. Unfortunately, purified F
could not be
obtained from the first two mutants because of impaired assembly and
oligomeric instability. This appears to be a general feature of
mutations at the
-Lys
locus. However, purified
F
was obtained from the
D261N/
R365W mutant, and
it provided valuable information about the noncatalytic sites.
From
the nucleotide-binding data described under ``Results'' we
can draw three conclusions. First, removal of the Asp carboxyl at
position -261 by the Asp
Asn mutation greatly reduces
noncatalytic site affinity for MgATP and MgADP (Fig. 2A and 3A), indicating that the
-Asp
carboxyl interacts directly with the Mg moiety of bound
Mg-nucleotide. This is consistent with the x-ray structure (Abrahams et al., 1994). Second, it is apparent that this interaction is
the primary determinant of the preference of noncatalytic sites for
Mg-nucleotides, and provides a large component of the overall binding
energy. From the K
values for MgATP and
MgADP in Table 2it is apparent that in combination residue
-Asp
and the Mg of the Mg-nucleotide provide about
3.2 kcal/mol of the MgATP binding energy, and that the corresponding
value for MgADP is about 2.5 kcal/mol. Third, since Asp is the residue
at position
-261 in wild-type, the natural ligands will be MgATP
and/or MgADP.
It was interesting that free ADP was bound with higher
affinity by the D261N/
R365W enzyme than by the
R365W
enzyme (Fig. 3B). A possible explanation is that free
ADP and the
-Asp
carboxyl in the noncatalytic sites
of the
R365W enzyme undergo mutual electrostatic repulsion, and
thus the Asp
Asn mutation can assist binding in this situation.
This was not the case, however, with free ATP, which bound overall with
relatively low affinity to both enzymes (compare Fig. 2B with Fig. 3B). The presence of two mutations in
the binding site in the
D261N/
R365W enzyme places limitations
on the interpretations of these findings.
For some time we have
propounded the view that F catalysis is not dependent upon
occupancy of the noncatalytic sites (Perlin et al., 1984; Wise
and Senior, 1985), although this idea has been challenged (Bullough et al., 1988; Milgrom et al., 1990, 1991; Allison et al., 1992; Harris, 1993). In a recent paper we provided
evidence showing that rapid rates of catalysis were achieved in
R365W F
under conditions where less than one
noncatalytic site was filled by nucleotide, on average, per enzyme
molecule (Weber et al., 1994). The data in this paper reaffirm
our conclusion, by demonstrating unequivocally that the
D261N/
R365W enzyme, with empty noncatalytic sites, still
hydrolyzes MgATP.
The D261N/
R365W mutant strain grew
normally on succinate plates and in limiting glucose medium, showing
that rates of oxidative phosphorylation in vivo were normal.
Thus, it might appear that oxidative phosphorylation occurs in this
strain without any requirement for the noncatalytic sites to be
occupied by nucleotide. It could be argued that the proton gradient
(
p) alters the behavior of the
D261N/
R365W noncatalytic
sites in membrane-bound enzyme, such that they are induced to bind
nucleotide, although this seems unlikely given the clear structural
rationale for the mutational effect. No doubt, however, the
D261N/
R365W mutant will be helpful in seeking answers to this
question, and also to the wider question of the possible regulatory or
modulating roles of the noncatalytic sites under the variety of growth
conditions encountered by E. coli in vivo.