(Received for publication, October 1, 1996, and in revised form, November 7, 1996)
From the Department of Biochemistry, University of Rochester Medical Center, Rochester, New York 14642
Three critical residues, -Lys-155,
-Asp-242, and
-Glu-181, situated close to the
-phosphate of
MgATP in F1-ATPase catalytic sites, were investigated. The
mutations
K155Q,
D242N, and
E181Q were each combined with the
Y331W mutation; the fluorescence signal of
-Trp-331 was used to
determine MgATP, MgADP, ATP, and ADP binding parameters for the three
catalytic sites of the enzyme. The quantitative contribution of side
chains to binding energy at all three catalytic sites was calculated.
The following conclusions were made. The major functional interaction
of
-Lys-155 is with the
-phosphate of MgATP and is of primary
importance at site 1 (the site of highest affinity) and site 2. Release
of MgATP during oxidative phosphorylation requires conformational
re-positioning of this residue. The major functional interaction of
-Asp-242 is with the magnesium of the magnesium nucleotide at site
1; it has little or no influence at site 2 or 3. In steady-state
turnover, the MgATP hydrolysis reaction occurs at site 1.
-Glu-181
contributes little to nucleotide binding; its major catalytic effect
derives apparently from a role in reaction chemistry per
se. This work also emphasizes that nucleotide binding
cooperativity shown by the three catalytic sites toward MgATP and MgADP
is absolutely dependent on the presence of magnesium.
The final step in oxidative phosphorylation, the synthesis of ATP
driven by a proton gradient, is carried out by the enzyme ATP synthase.
It is abundant in mitochondria, chloroplasts, and bacteria; in
bacteria, it is also used to build a transmembrane proton gradient,
using ATP hydrolysis as the energy source. The enzyme consists of the
membrane-embedded F0-sector, containing the
proton-conducting pathway, and the membrane-extrinsic
F1-sector, containing the nucleotide-binding sites.
Purified, soluble F1 retains ATPase activity and is
extensively used as an experimental model. It shows the subunit
composition 3
3
(for reviews, see
Refs. 1-3). X-ray crystallography of bovine mitochondrial F1 demonstrated that the three
- and three
-subunits
are alternately arranged in a hexagon, with
in the center. The
three catalytic sites are made up predominantly by
-subunit
residues, with two side chains from
-subunits also in close
proximity to the bound nucleotide (4).
F1 shows strong positive catalytic cooperativity. If just one of the three catalytic sites becomes occupied by MgATP ("unisite catalysis"), hydrolysis proceeds slowly, and the products MgADP and Pi are released very slowly (5, 6). In a molecule in which two sites have bound MgATP ("bisite catalysis"), hydrolysis proceeds, but release of products is slow, so that net turnover remains slow (7). Only when all three catalytic sites in each molecule bind MgATP does rapid, physiological ATPase turnover ensue (8). Under conditions of rapid turnover, the vast majority of molecules in the solution exist at any given point in time in a state in which all three catalytic sites are occupied, two by MgADP and one by MgATP (7).
Deduction of the catalytic properties of F1 has relied heavily on development of a true equilibrium binding technique for analysis of MgATP binding. It had been shown that in active, soluble, purified F1, all three catalytic sites bind the non-hydrolyzable nucleotide analogs MgAMP-PNP1 and Mg-lin-benzo-ADP (9, 10), and these reports demonstrated pronounced binding cooperativity among the three sites. For analysis of MgAMP-PNP binding, it was possible to use radioactive nucleotide in conjunction with the centrifuge column technique because significant loss did not occur during centrifugation. For analysis of Mg-lin-benzo-ADP binding, the intrinsic fluorescence signal of the analog itself could be used. Neither of these approaches was viable for measurement of MgATP binding. For MgATP binding, the method of choice has proved to be tryptophan fluorescence spectroscopy using specifically engineered tryptophan residues placed strategically in the catalytic sites as reporter probes. This technique has demonstrated that all three sites bind MgATP, with marked binding cooperativity being evident (8, 11). Parallel results were obtained with the slowly hydrolyzed analog Mg-TNP-ATP (12). Magnesium is critical for catalytic site binding cooperativity, and in its absence, all three sites bound ATP with the same affinity (Kd(ATP) = 71 µM) (11). That is to say, the catalytic sites behave symmetrically toward nucleotides in the absence of magnesium.
Site-directed mutagenesis is a valuable approach for studies of enzyme
mechanism and has already been applied advantageously to studies of
F1 catalytic sites (1-3). Three residues that have proved
to be critical for catalysis are -Lys-155,
-Asp-242, and
-Glu-181 (13-18).
-Lys-155 is located in the Homology A (P-loop) motif (19), and previous mutagenesis experiments indicated that
-Lys-155 interacts with the
-phosphate of MgATP, contributing necessary binding energy to drive catalysis (13, 14).
-Asp-242 is
located in the Homology B motif (19), and mutagenesis suggested that
the critical activity of this residue derives from its role as a ligand
for magnesium in MgATP (13). Several laboratories have studied
-Glu-181, and there is general agreement that a carboxyl group at
this position is critical (13, 16-18). Our laboratory reported that
the mutation
E181Q had a dramatic effect on the reaction equilibrium
and rate constants in unisite catalysis by destabilizing the reaction
transition state (13). Thus,
-Glu-181 has been considered to be
involved in the catalytic reaction step.
The x-ray structure of bovine mitochondrial F1 (4) revealed
that previous proposals regarding the locations of -Lys-155,
-Asp-242, and
-Glu-181 were correct and that previous deductions about the roles of these residues in catalysis were well founded. Fig. 1A shows the position of each in the
catalytic site. In addition, it may be noted that the discovery of a
water molecule situated between
-Glu-181 and the
-phosphate of
MgATP (see Fig. 1A) led to the suggestion (4) that
-Glu-181 might act as a catalytic base, activating the water for
nucleophilic attack on the
-phosphate.
In previous mutagenesis studies of -Lys-155,
-Asp-242, and
-Glu-181, the catalytic and nucleotide binding parameters reported were restricted to catalytic site 1 (the site of highest affinity) because only "unisite" techniques could be used. The tryptophan fluorescence technique had not yet been developed, and so nucleotide binding parameters for catalytic sites 2 and 3 were not accessible. Therefore, to further investigate the molecular role of these three
critical residues, we combined each of the inhibitory mutations,
K155Q,
D242N, and
E181Q, with the
Y331W mutation and
determined nucleotide binding parameters at all three catalytic sites.
As shown in Fig. 1B, in wild-type enzyme,
-Tyr-331 makes
van der Waals contact with the adenine ring of bound nucleotide in the catalytic sites. When Trp is substituted at residue
331, it shows a
large fluorescence signal that is completely quenched on binding of
nucleotide (8). Purified F1 was obtained from each of the mutants
K155Q/
Y331W,
D242N/
Y331W, and
E181Q/
Y331W,
and binding parameters for MgATP, MgADP, ATP, and ADP at each of the
three catalytic sites were determined using the
-Trp-331
fluorescence signal.
The K155Q mutation was moved from plasmid
pDP33 (20) into plasmid pSWM4 (which contains the
Y331W mutation
(8)) by replacing the 1.0-kilobase Bst1107I fragment in
pSWM4 with the corresponding fragment from pDP33. The resultant plasmid
was named pSWM31. The presence of both mutations in pSWM31 was
confirmed by DNA sequencing. Strain JP17 (21), which contains a large deletion in the chromosome
-subunit gene, was transformed with plasmid pSWM31 to yield strain SWM31.
The
same procedure as described above was followed, with the exception that
the 1.0-kilobase Bst1107I fragment carrying the E181Q
mutation originated from plasmid pDP36 (20). The mutant strain
(pSWM32/JP17) was named SWM32.
The
2.5-kilobase HindIII-KpnI fragment from pSWM4,
containing the entire -subunit gene with the
Y331W mutation, was
ligated into M13mp18, and mutagenesis was performed as described
previously (10). The mutagenic oligonucleotide was
TGCTGTTCGTT
ACAACATC, the underlined base change changing
residue
242 from Asp to Asn (GAC to AAC) and introducing a new
HpaI site. The 2.2-kilobase NheI-KpnI
fragment was transferred from mutant phage into pSWM4, generating the
new plasmid pSWM36. DNA sequencing was performed to show that the
-subunit gene in pSWM36 contained both
D242N and
Y331W
mutations and no undesired mutations. pSWM36 was introduced into
strain JP17, yielding strain SWM36.
Growth yield analysis in limiting (3 mM) glucose liquid medium and growth tests on solid succinate medium were performed as described (22).
Enzyme Purification and CharacterizationF1 was
purified from each of the mutant strains described above and from
wild-type strain SWM1 (23) according to Weber et al. (10).
ATPase assays were performed as described by Weber et al.
(11), except that the ATPase activities shown in Fig. 8 were measured
at 23 °C and pH 8.0. Procedures for SDS-gel electrophoresis and
protein assay were as described previously (10).
Fluorescence Measurements
For studies of binding of nucleotides, F1 was pre-equilibrated in buffer (50 mM Tris/SO4, pH 8.0) by dilution to ~8 mg/ml and passage of 100-µl aliquots consecutively through two 1-ml Sephadex G-50 centrifuge columns. In previous work (8, 11), this procedure was seen to effectively remove catalytic site-bound nucleotide. Fluorescence experiments were carried out using a Spex Fluorolog 2 spectrofluorometer at 23 °C. Final enzyme concentration in the cuvette was 100-200 nM. For MgADP binding experiments, the buffer (50 mM Tris/SO4, pH 8.0) contained 2.5 mM MgSO4, and ADP was added as indicated. Titrations with MgATP were performed by adding ATP and MgSO4 in a constant ratio of 2.5:1. MgATP and MgADP concentrations were calculated according to Ref. 24. For ADP and ATP binding experiments, the 50 mM Tris/SO4 pH 8.0 buffer contained 0.5 mM EDTA, and ADP or ATP was added as indicated. Excitation and emission wavelengths were 295 and 360 nm, respectively. Background signals (buffer, Raman scatter) were subtracted; inner filter and volume effects were corrected for by conducting parallel titrations with wild-type F1. Evaluation of the data was achieved using DATA-MAX software; calculation of nucleotide binding parameters was performed according to Ref. 8 using the equations given in the figure legends.
In Table
I, the effects of the double mutations are presented and
compared with data for each of the mutations when present singly. In
accordance with the results obtained for the single mutations K155Q,
E181Q, and
D242N (13), none of the double mutants was able to
grow on succinate plates, nor did the growth yields in medium
containing limiting (3 mM) glucose exceed that of the
negative Unc
control. Thus, all three mutations,
K155Q,
E181Q, and
D242N, abolish ATP synthase activity
in vivo when present in combination with the
Y331W
mutation. As reported previously (8), the
Y331W mutation alone had
only a minor effect on ATP synthase in vivo.
|
As judged from the Sephacryl S300 elution profile in the
final step of purification and from SDS-gel electrophoresis, purified F1 from all three double mutants showed the same molecular
size and subunit composition as wild-type F1. The specific
ATPase activity of purified F1 was determined and in each
case was very low indeed (Table I, fourth column). In the case of the
K155Q/
Y331W and
D242N/
Y331W enzymes, the activities were
similar to those seen earlier (13) for the
K155Q and
D242N single
mutant enzymes. For the
E181Q/
Y331W double mutant enzyme, no
ATPase activity was observed (limit of detection was
0.001 units/mg),
whereas the
E181Q single mutant F1 preparation showed
very low but detectable activity in previous work (13). The reason for
this difference is not clear; nevertheless, the data of Table I show
clearly that each of the mutations under study retains its potent
inhibitory effect on catalysis in purified enzyme when present with the
Y331W mutation. It may be recalled that the
Y331W mutation alone
reduces kcat by ~50%, but has no effect on
kcat/Km in purified F1 (8).
Tryptophan fluorescence spectra of purified
F1 from all three double mutants were very similar to that
of Y331W mutant F1 (see Fig. 2 in Ref. 8), and in each
case, the
W331 fluorescence signal was quenched virtually completely
upon addition of saturating nucleotide, providing an ideal tool to
determine effects of each of the three inhibitory mutations on
catalytic site nucleotide binding parameters. The similarity of the
fluorescence spectra indicates normal folding of the
-subunits in
the mutant enzymes.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
Nucleotide Binding to
Titration curves for MgATP and MgADP binding are
presented in Fig. 2 (A and B,
respectively). Dissociation constants were determined by fitting
theoretical binding curves to the experimental data by nonlinear
least-squares regression analysis. The lines in Fig. 2A
represent fits to the MgATP binding data using a model assuming three
independent binding sites per F1 (see Fig. 2A
legend). As determined previously (8, 11), this model provides the optimal fit with the Y331W enzyme. The calculated dissociation constants are given in Table II. Two conclusions are
immediately apparent: (i) instead of three catalytic sites each of very
widely different affinity, as in
Y331W F1, the
K155Q/
Y331W enzyme has Kd2 very
similar to Kd3; and (ii) the
K155Q mutation strongly reduces binding affinity for MgATP at catalytic sites
1 and 2, but has a much smaller effect on site 3. It should be noted
that in
Y331W F1, only a limit value of
50
nM can be assigned to Kd1
from the fluorescence data because catalytic site 1 was already filled
at the lowest concentration of MgATP used (170 nM).
Experiments with wild-type F1 using radioactive MgATP and
unisite techniques yielded a much lower value for
Kd1 of 0.2-0.4 nM (13, 25). Therefore,
the effects of the
K155Q mutation on Kd1 are
much larger than is apparent from the data in Table II, amounting to a
reduction of 3 orders of magnitude. Previous determination of
Kd1(MgATP) for the
K155Q single mutant enzyme
using unisite techniques gave values of 0.3-0.4 µM (13,
14), in good agreement with the value of 0.5 µM for the
K155Q/
Y331W mutant reported in Table II.
|
Fig. 2B shows titration curves for MgADP binding. The lines
represent fits to a model assuming two classes of binding sites (see
Fig. 2B legend). As noted previously, this model provides the optimal fit for MgADP binding to Y331W F1 (8). As
Table II shows, the
K155Q mutation had no significant effect on
calculated MgADP binding affinity at any of the catalytic sites. The
difference between the two curves in Fig. 2B is due to
different N values for the two classes of binding sites.
Whereas for
Y331W F1, the best fit is obtained with 1.1 sites of higher affinity and 1.4 sites of lower affinity, for
K155Q/
Y331W F1, the corresponding N values
are 0.4 and 2.3, respectively. An explanation for the occurrence of
non-integer binding stoichiometries is not apparent. However, even if
the model assumed for calculation of binding parameters is not fully
correct, still it is apparent from Fig. 2B that, at most,
the
K155Q mutation perturbs MgADP binding only at site 1, and then
not to a major degree.
Titration curves for ATP and ADP (i.e. absence of magnesium
and presence of 0.5 mM EDTA) are presented in Fig.
3 (A and B, respectively). In all
cases, a model assuming a single class of binding site (solid
lines) gave an adequate fit (see Fig. 3 legend). For the Y331W
enzyme, the number of sites (N) = 2.9 for ATP and 2.7 for
ADP; for
K155Q/
Y331W F1, N = 3.0 for
ATP and 2.9 for ADP. The calculated binding affinities are given in
Table II. It is apparent that the binding affinity for ATP is decreased considerably by the
K155Q mutation and that the binding affinity for
ADP is reduced, but to lesser extent.
![]() |
(Eq. 3) |
Together, the data presented in Figs. 2 and 3 show that the lysine
residue in position 155 of F1 catalytic sites interacts primarily with the
-phosphate of the nucleotide, as has been proposed earlier (14). The
K155Q mutation strongly reduced binding
affinities for MgATP and ATP, but affected ADP and MgADP binding much
less. Moreover, the three catalytic sites are not the same:
-Lys-155
contributes far more to binding of MgATP at sites 1 and 2 than it does
at site 3. This difference is important for the release of MgATP during
oxidative phosphorylation, as discussed below.
Titration curves for binding of MgATP and MgADP to
E181Q/
Y331W F1 are displayed in Fig. 4
(A and B, respectively). As described above, the
MgATP data were fitted assuming a model with three independent binding
sites (Fig. 4A), and calculated Kd values
are given in Table II. It is evident that the
E181Q mutation has no
significant influence on binding of MgATP at catalytic sites 2 and 3. Kd1 for
E181Q/
Y331W F1
could not be accurately calculated because catalytic site 1 was already
filled at the lowest concentration of MgATP used (170 nM).
Inspection of the binding curves in Fig. 4A indicates little
effect of the
E181Q mutation on Kd1.
However, it should be noted that previous unisite experiments indicated
a Kd1 of 40 nM for the
E181Q single mutant enzyme (Ref. 13; see also Ref. 16).
For MgADP binding in Fig. 4B, a model assuming two classes
of binding sites (as described above) gave a good fit to the
E181Q/
Y331W data, with 0.5 sites of higher affinity and 1.9 sites
of lower affinity. The calculated
Kd(MgADP) values (Table II) indicate
that there was no effect of the
E181Q mutation on MgADP binding
affinity at any of the catalytic sites. The difference between the
E181Q/
Y331W and
Y331W curves in Fig. 4B is again due to differences in binding stoichiometry (N values) at
site 1 as compared with sites 2 and 3 (see above), and the same
comments apply.
Curves for binding of ATP and ADP to E181Q/
Y331W F1
in the absence of magnesium are reported in Fig. 5
(A and B, respectively). ATP bound to 2.6 sites,
each with Kd of 20 µM, and ADP bound
to 2.8 sites, each with Kd of 30 µM.
Thus, both nucleotides were bound with slightly higher affinity in the
E181Q/
Y331W mutant than in the parental
Y331W enzyme (Table
II). Elimination of the negative charge of the
-Glu-181 residue
could remove a repulsion toward the phosphates when no magnesium is
present for compensation of their negative charge.
Overall, the data show that -Glu-181 is not primarily involved in
nucleotide binding, especially at catalytic sites 2 and 3. At catalytic
site 1, it makes no contribution to MgADP binding and, at most, a small
contribution to MgATP binding.
Binding curves obtained by titration of
D242N/
Y331W F1 with MgATP and MgADP are shown in Fig.
6 (A and B, respectively). For
MgATP, the model assuming three independent binding sites gave a good
fit to the data, and calculated values for dissociation constants are
given in Table II. The highest affinity catalytic site 1 was most
affected by the
D242N mutation, with
Kd1 being increased to 0.3 µM. Previous work using unisite techniques had given a
similar value of 0.28 µM for
Kd1 at pH 8.0 in
D242N single mutant
F1.2 In contrast, at sites 2 and 3, significant changes in Kd(MgATP) did not occur (Table II). A similar result was obtained from the MgADP
binding curves (Fig. 6B). Here,
Kd(MgADP) = 12 µM at all
sites (Table II) (N = 2.5). Thus, the MgADP-binding site of higher affinity had effectively disappeared in the
D242N/
Y331W enzyme.
Binding of ATP and ADP to D242N/
Y331W F1 in the
absence of magnesium is shown in Fig. 7 (A
and B, respectively). In both cases, a model assuming a
single class of binding site gave a satisfactory fit, and calculated
dissociation constants are shown in Table II. ATP and ADP were both
bound with slightly higher affinity in the
D242N/
Y331W mutant
than in the
Y331W parent enzyme. In
D242N/
Y331W
F1, N = 2.7 with ATP and 2.7 with ADP.
From its location in the catalytic site, as revealed in the x-ray
structure (Fig. 1A), -Asp-242 is positioned to be one of the coordinating ligands for the magnesium ion in MgATP (4). Such a
role was adumbrated by biochemical studies of the
D242N mutant (13)
and has been proposed for the analogous Asp residue in the Homology B
sequence in other enzymes. Data in this paper confirm that the carboxyl
side chain of
-Asp-242 does bind the magnesium of MgATP and MgADP;
moreover, they show that this role is limited to catalytic site 1. MgATP and MgADP binding at catalytic sites 2 and 3 was not affected by
the
D242N mutation. Thus,
-Asp-242 is an important contributor to
the high binding affinity of catalytic site 1 for MgATP, and it is very
reasonable to propose that release of MgATP during oxidative
phosphorylation involves disengagement of the bond between the Asp-242
side chain and the magnesium ion.
-Asp-242 also contributes to
binding of MgADP at catalytic site 1, to a lesser extent than with
MgATP.
The behavior seen with ADP and ATP in the D242N/
Y331W mutant
supports these conclusions. With both nucleotides, all three catalytic
sites show the same affinity, with no cooperativity evident, and the
dissociation constant is similar to that of sites 2 and 3 for MgATP or
to that of sites 1-3 for MgADP (Table II). Binding of ATP and ADP in
the
D242N/
Y331W mutant is actually slightly tighter than in the
Y331W parental enzyme (22 versus 71 µM for
ATP and 11 versus 83 µM for ADP). The missing
negative charge of
-Asp-242 may lessen repulsion toward the negative
phosphates of the nucleotides in the absence of magnesium.
The results described above
showed that -Asp-242 plays a critical role in binding of MgATP
specifically at catalytic site 1, and we wished to find whether this
role is correlated with effects on steady-state catalysis. Excess
magnesium ion inhibits ATPase activity in E. coli
F1 (9), and we used this property to investigate this
question. ATPase activity of
D242N/
Y331W double mutant
F1 was assayed at a constant ATP concentration (5 mM) in the presence of increasing magnesium ion
concentration (Fig. 8). In the wild type and
Y331W,
an ATP/magnesium ratio of 2.5:1 proved optimal for activity as
previously established, and increasing concentrations of magnesium ion
led to significant inhibition. In contrast, ATPase activity in the
D242N/
Y331W mutant was not inhibited by magnesium even at 25 mM. Thus, the mutant had lower affinity for inhibitory
magnesium cation. Control experiments showed that binding of MgATP to
all three sites was retained under saturating conditions in the
presence of 25 mM magnesium ion in both
D242N/
Y331W
and
Y331W enzymes. These results confirm the role of
-Asp-242 in
catalysis through its role in liganding magnesium of the substrate
MgATP, and they emphasize that catalytic site 1 is the catalytic site
at which steady-state catalysis occurs.
The aim of this work was to study the molecular role
of -Lys-155,
-Asp-242, and
-Glu-181 in catalytic sites of
F1-ATPase. The first two are located in the P-loop and
Homology B sequences, respectively. All three are located close to the
-phosphate of bound MgATP (4), and previous mutagenesis experiments
from this and other laboratories have shown that all three are critical for both ATP synthesis and ATP hydrolysis. Application of unisite techniques has revealed a wealth of information regarding the molecular
role of these residues at catalytic site 1 (highest affinity site), but
there is not yet comparable information regarding catalytic sites 2 and
3. We recently introduced the technique of site-directed tryptophan
fluorescence spectroscopy to determine nucleotide occupancy and binding
parameters in catalytic sites of F1 under true equilibrium
conditions (8,11). Here, we applied this technique to determine
functional effects of the mutations
K155Q,
D242N, and
E181Q at
all three catalytic sites.
We found that -Lys-155 is particularly
important for MgATP binding: it has its major effects at catalytic
sites 1 and 2 and a small effect at catalytic site 3 (the site of
lowest affinity) (Figs. 2 and 3 and Table II). The contributions of
-Lys-155 to overall MgATP binding energy are as follows: site 1, 3.9 kcal/mol (calculated from data in Refs. 13, 14, and 25); site 2, 3.4 kcal/mol (this work); and site 3, 1.1 kcal/mol (this work). Lack of
strong interaction between
-Lys-155 and MgATP at catalytic site 3 appears to be the major reason for the relatively low affinity of this
site. On the other hand,
-Lys-155 did not provide binding energy for
MgADP binding at any site (Table II). The primary functional interaction of
-Lys-155 is therefore with the
-phosphate of bound
MgATP.
In the x-ray structure of F1 (4), only two catalytic sites
are filled, one with MgAMP-PNP and one with MgADP. It is not known
which corresponds to site 1 (highest affinity) and which to site 2. However, it is valuable to note the spatial relationship of -Lys-155
to the nucleotide in both cases. In the MgAMP-PNP-containing site (Fig.
1A), the distance from the
-amino group of
-Lys-155 to
the nearest
-phosphate oxygen of the nucleoside triphosphate is 2.7 Å, and that to the nearest
-phosphate oxygen is 3.3 Å, whereas the
distance to the magnesium ion is 5.4 Å. In the MgADP-containing site,
corresponding distances are 2.7 Å (to the nearest
-phosphate oxygen) and 5.2 Å (to magnesium).
It has been proposed that in oxidative phosphorylation, the proton
gradient provides necessary energy to release MgATP from F1
by overcoming the barrier for changing binding affinity at the site
where MgATP is synthesized from tight to loose (26). Table II shows
that upon introduction of the K155Q mutation, the binding affinity
for MgATP at catalytic site 1 was drastically reduced. Conformational
changes necessary for proton gradient-induced release of MgATP during
ATP synthesis must therefore involve movement of
-Lys-155 away from
the
-phosphate. For MgATP hydrolysis, the critical role of
-Lys-155 must derive from its immobilization and orientation of the
nucleotide, at least in part, and other mechanisms may also be
operative.
Our studies on -Lys-155 are also germane to the question of what
mechanism determines binding cooperativity among the three catalytic
sites. It was recently suggested that structural asymmetry of
F1 caused by the
-subunit is responsible for the
generation of the highest affinity catalytic site (27). Since, in
intact F1, binding of ATP or TNP-ATP (in the absence of
magnesium) occurs with equal or very similar affinity at all three
catalytic sites (11, 12), and we show further in Table II of this paper
that binding of ADP (in the absence of magnesium) also occurs with equal affinity at all three sites, it is evident that while the
-subunit certainly is necessary for binding cooperativity to be
displayed, it is not sufficient. Data in this paper actually substantiate this argument at the molecular level. When MgATP occupies
the catalytic sites,
-Lys-155 must occupy a different position in
relation to the nucleotide in each of site 1, site 2, and site 3 because the
K155Q mutation affects the three sites differentially
(Table II). In contrast, when ATP occupies the catalytic sites,
-Lys-155 appears to occupy the same position in relation to the
nucleotide in all three sites since the
K155Q mutation affects all
three sites equally.
From its location in the catalytic site (Fig.
1A) and by analogy with other enzymes, one of the
coordinating ligands for the magnesium of MgATP is predicted to be
-Asp-242, through an intervening hydrogen-bonded water. This work
establishes that
-Asp-242 is one of the magnesium ligands and,
moreover, that this role is realized predominantly at catalytic site 1. The binding energy contributed by
-Asp-242 to MgATP binding at site
1 is 3.0 kcal/mol, and the values at sites 2 and 3 are 1 and 0 kcal/mol, respectively. The result for site 3 was not unexpected, as
there is no significant difference between the binding affinity for
MgATP at site 3 and for free ATP at any of the three sites in
Y331W
F1 (Table II). The binding energy contributed by
-Asp-242 to MgADP binding at sites 1-3 is 2.7, 0, and 0 kcal/mol,
respectively. Distances between the magnesium and
-Asp-242 carboxyl
oxygens in the x-ray structure of the MgAMP-PNP-containing site (Fig.
1A) are 3.9 and 4.1 Å. The closest contacts of magnesium
are with oxygens of the
-phosphate (2.2 Å) and
-phosphate (2.5 Å) and the hydroxyl of
-Thr-156 (2.3 Å). In the MgADP-containing
site, the two
-Asp-242 carboxyl oxygens are 4.3 Å from the
magnesium. The closest contacts of magnesium are with oxygens of the
-phosphate (2.3 Å) and the hydroxyl of
-Thr-156 (2.3 Å).
Removal of the carboxyl side chain at position 242 not only
decreased the affinity at site 1 for both MgATP and MgADP, it also
abolished the inhibitory effect of magnesium ion on ATPase activity
(Fig. 8). Taken together with the fact that the
D242N mutation
reduced nucleotide binding affinity primarily at site 1, this
emphasizes that site 1 is where hydrolysis occurs in steady-state catalysis.
As described in the Introduction, site-directed
mutagenesis studies have shown that -Glu-181 plays a critical role
in catalysis in F1-ATPase and that the carboxyl group is of
paramount importance. Abrahams et al. (4) suggested that
-Glu-181 could act as a catalytic base by activating a water
molecule to perform in-line nucleophilic attack on the
-phosphate of
MgATP. In the x-ray structure of the MgAMP-PNP-containing site (Fig.
1A), this water is situated 4.1 Å from the
-phosphorus
atom; the nearest carboxyl oxygen of
-Glu-181 is 5.1 Å from the
-phosphorus atom. In the MgADP-containing site, a similarly
positioned water is absent, and the nearest carboxyl oxygen of
-Glu-181 is 7.0 Å from the
-phosphorus atom. The results
presented in this study establish that the mutation
E181Q had no
influence on the binding affinity for MgATP at catalytic sites 2 and 3 or on the binding of MgADP, ATP, or ADP at site 1, 2, or 3. There may
be some effect on MgATP binding at site 1 (see "Results"), but
taken overall,
-Glu-181, although located in the phosphate-binding
pocket, clearly contributes less to nucleotide binding or positioning
than either of the other two residues studied here. Thus, its critical
role in catalysis most likely derives from a different
source.3
Information obtained so far from mutagenesis of F1 in
several laboratories implicates -Glu-181 in reaction chemistry, but does not yet point to any specific mechanism. Within different nucleoside triphosphate-hydrolyzing enzymes, a variety of answers to
the question of whether a general base residue is present have been
proposed. For p21ras, it was suggested initially that Gln-61 is
the catalytic base, but more recent work suggests that in this protein,
the
-phosphate of GTP itself acts as the base, leaving a stabilizing
and orienting role for Gln-61 (28-31). Sato et al. (32)
suggest that Asp-133 is the catalytic carboxylate that activates a
water molecule for the attack of the
-phosphate in SecA protein of
E. coli. Lys-71 plays an important role in bovine Hsc70
protein, and ATPase activity was abolished in mutants K71E, K71M, and
K71A. Crystals obtained from these mutants contain bound ATP, and
participation of Lys-71 in catalysis as a proton acceptor has been
proposed (33). In myosin, where no obvious catalytic base candidate is
present in the catalytic site, a highly conserved serine residue
associated with the
-phosphate-binding pocket may function as an
exchanger of protons between the phosphate and the attacking water
molecule (34). Thus, it would seem that the presence of a catalytic
base side chain is not a sine qua non for rapid nucleoside
triphosphatase activity, and before concluding that
-Glu-181 is
acting as catalytic base, further work will be needed. It may be
pertinent that the unisite MgATP hydrolysis rate remained unchanged at
pH 5.5-9.5 (35).
-Glu-181 may stereochemically orient and polarize
the attacking water without net proton abstraction.