From the Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642
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ABSTRACT |
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Escherichia coli
F1-ATPase from mutant F1-ATPase is the catalytic sector of the enzyme
F1F0-ATP synthase, responsible for ATP
synthesis by oxidative phosphorylation in mitochondria, chloroplasts,
and bacteria and also for ATP-driven proton pumping in bacteria. It
consists of five different subunits in the stoichiometry
To understand the mechanism of ATP synthesis and hydrolysis in
F1, the manner in which the catalytic transition state is
attained and stabilized by catalytic site residues must be established. We have previously studied the catalytic transition state using techniques of "unisite catalysis." Unisite catalysis is the term used to describe the single turnover of ATP hydrolysis that occurs under conditions where F1 is present in excess over
substrate MgATP (6). Complete characterization of all the rate and
equilibrium constants of unisite catalysis allows one to construct
Gibbs free energy diagrams for the entire catalytic pathway, including
the catalytic transition state (7). Construction of difference energy
diagrams for mutant versus wild-type enzymes then readily reveals situations where destabilization of the catalytic transition state has been brought about by mutagenesis (8, 9). With this
technique, the roles of several critical catalytic site residues were
defined in our laboratory (7-10). The x-ray structures have since
confirmed their proximity to the bound substrate in the catalytic sites
and revealed their actual locations in exquisite detail.
However, unisite catalysis assays require significant quantities of
both enzyme and radioactive isotope and are time-consuming. An
alternative approach to study the transition state that overcomes these
problems would be of great value. Earlier, Vignais and colleagues (11-13) had shown that the MgADP-fluoroaluminate complex was a potent
inhibitor of mitochondrial and Escherichia coli
F1-ATPase. At that time, it was already recognized that the
MgADP-fluoroaluminate complex bound in catalytic sites of ATP- or
GTP-hydrolyzing enzymes was most likely mimicking the catalytic
transition state, and the data reported by Vignais and colleagues
strongly supported the view that this was also the case for
F1. Subsequent publication of x-ray crystallography
structures of a variety of ATPase and GTPase enzymes with
MgADP-fluoroaluminate bound in the catalytic sites (14-22) has
established definitively that MgADP-fluoroaluminate complex is a
catalytic transition state
analog.1
When the Preparation of Enzymes--
Wild-type F1 was from
strain SWM1 (25), Inhibition of F1-ATPase by
MgADP-fluoroaluminate--
F1 (0.24 mg/ml) was
preincubated at room temperature in 50 mM
Tris/SO4, pH 8.0, with or without 2.5 mM MgADP,
AlCl3, and NaF. Aliquots (100 µl) were removed, and
ATPase activity was assayed in a total volume of 1 ml containing 50 mM Tris/SO4, pH 8.5, 10 mM NaATP,
and 4 mM MgCl2 at room temperature for 2-5
min, at which time the reaction was stopped by the addition of 1 ml of
10% sodium dodecyl sulfate. Pi release was linear with
time and was measured as in Ref. 29.
Inhibition of F1-ATPase by DCCD--
This was
carried out as described by Weber et al. (27) using 500 µM DCCD. The enzyme was assayed as above.
Fluorescence Measurements--
Fluorescence measurements were
made at room temperature in 50 mM Tris/SO4, pH
8.0. A SPEX Fluorolog 2 or Aminco-Bowman 2 spectrofluorometer was used.
The excitation wavelength was 295 nm, and fluorescence emission at 360 nm was used as the signal (23, 27). For MgADP titration in absence of
fluoroaluminate, the buffer contained 2.5 mM
MgSO4, and NaADP was added. For MgADP titration in presence of fluoroaluminate, the buffer contained 2.5 mM
MgSO4, 0.5 mM AlCl3, and 5 mM NaF, and NaADP was added. For ADP titration (absence of
Mg2+), the buffer contained 0.5 mM EDTA, and
NaADP was added. Enzyme was preincubated 60 min at room temperature
before fluorescence signals were measured to allow full inhibition by
fluoroaluminate to be attained. Background signals (buffer) were
subtracted, and inner filter and volume effects were corrected by
performing parallel titrations with wild-type F1.
Nucleotide binding parameters were analyzed by fitting theoretical
curves to the measured data points assuming theoretical models with
one, two, or three types of binding sites as described in detail
previously (24). MgADP concentrations were calculated using the
stability constant of 78 µM from Ref. 30.
Inhibition of ATP Hydrolysis Activity of Conditions for potent inhibition of mitochondrial and wild-type
E. coli F1-ATPase by MgADP in combination with
AlCl3 and NaF, which together form fluoroaluminate
complexes, have been documented by Vignais and colleagues (11-13).
Here it was necessary to confirm that the Y331W was potently inhibited by
fluoroaluminate plus MgADP but not by MgADP alone.
-Trp-331
fluorescence was used to measure MgADP binding to catalytic sites.
Fluoroaluminate induced a very large increase in MgADP binding affinity
at catalytic site one, a smaller increase at site two, and no effect at
site three. Mutation of either of the critical catalytic site residues
-Lys-155 or
-Glu-181 to Gln abolished the effects of
fluoroaluminate on MgADP binding. The results indicate that the
MgADP-fluoroaluminate complex is a transition state analog and
independently demonstrate that residues
-Lys-155 and (particularly)
-Glu-181 are important for generation and stabilization of the
catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme,
with 1% residual steady-state ATPase, showed normal transition state
formation as judged by fluoroaluminate-induced MgADP
binding affinity changes, consistent with a proposed mechanism by
which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable
for future transition state studies of F1-ATPase.
INTRODUCTION
Top
Abstract
Introduction
References
3
3
and contains three catalytic
nucleotide-binding sites whose structures have been defined by x-ray
crystallography at 2.8 Å resolution (1, 2). Each catalytic site lies
at an interface between an
- and
-subunit of the enzyme, with
most of the ligands between the nucleotide and the protein being
provided by side chains from the
-subunit. Rotation of the
-subunit within the
3
3 hexagon has
been demonstrated during MgATP hydrolysis (3), indicating that the
three catalytic sites participate in catalysis in sequential fashion.
Functional characterization of catalysis has been facilitated by
mutagenesis of critical catalytic site residues and also by
introduction of specifically engineered Trp residues into the catalytic
sites. The latter technique has enabled determination of catalytic site
nucleotide binding parameters in wild-type and mutant enzymes under a
wide range of conditions (for recent reviews see Refs. 4 and 5).
Y331W mutation is engineered into catalytic sites of
E. coli F1, the fluorescence of residue
-Trp-331 provides a sensitive and specific probe of catalytic site
nucleotide binding (23). The fluorescence signal can readily be
monitored at low enzyme concentration, and because the aromatic ring of
residue
-331 stacks against the adenine ring of bound nucleotide
(24), the fluorescence signal is totally quenched upon binding of
adenine nucleotide in the catalytic sites. We therefore hypothesized
that in
Y331W mutant enzyme (which, it should be stated, shows
normal catalytic behavior in ATP synthesis and hydrolysis), the
MgADP-fluoroaluminate complex should be readily detected as a very high
affinity species by fluorescence titration. In this paper, we use this
approach to measure the effects of fluoroaluminate on MgADP binding to catalytic sites in normal, mutant, and
DCCD2-inhibited enzyme.
EXPERIMENTAL PROCEDURES
Y331W mutant F1 was from strain SWM4
(23),
K155Q/
Y331W mutant F1 was from strain pSWM31
(24), and
E181Q/
Y331W mutant F1 was from strain
pSWM32 (24). Purification of F1 was as in Ref. 26. Before
use the enzymes were passed twice through 1-ml centrifuge columns of
Sephadex G-50 in 50 mM Tris/SO4, pH 8.0, as
described (27), which was shown to effectively remove catalytic
site-bound nucleotide ("native" enzyme). Nucleotide-depleted
F1 (depleted of both catalytic and noncatalytic site-bound
nucleotide) was prepared as described by Senior et al. (28).
Before use, it was passed once through a centrifuge column equilibrated
with 50 mM Tris/SO4, pH 8.0 (as above), and
then EDTA was added to a final concentration of 2 mM. Then,
after 1 h at room temperature, the enzyme was passed through a
centrifuge column equilibrated in 50 mM
Tris/SO4, pH 8.0.
RESULTS
Y331W F1 by
MgADP-fluoroaluminate Complex but Not by MgADP Alone
Y331W mutant enzyme was
also subject to the same inhibition. Table
I shows the data. It is seen that
complete inhibition was achieved in the presence of MgADP,
AlCl3, and NaF and that all three of these components were
necessary. It should also be noted that preincubation with MgADP alone,
at 2.5 mM concentration, had no significant inhibitory
effect. The small reduction in activity seen (8%) is readily explained
as due to competitive inhibition by MgADP carried over from the
preincubation into the enzyme assay. We emphasize that our ATPase
assays are carried out under conditions where free [Mg2+]
is minimal, by use of an ATP/Mg2+ concentration ratio of
10:4, which minimizes any potential inhibition by MgADP (28).
Inhibition of ATPase activity of Y331W F1 by
MgADP-fluoroaluminate complex
Y331W mutant F1 was passed through two centrifuge columns in
50 mM Tris/SO4, pH 8.0, as described under
"Experimental Procedures" and then preincubated for 60 min at room
temperature in 50 mM Tris/SO4, pH 8.0, with
additions of MgADP (2.5 mM), AlCl3 (1 mM), and NaF (5 mM) as indicated. ATPase
activity was assayed as described under "Experimental Procedures."
Further experiments showed that for complete inhibition the minimal concentrations of AlCl3 and NaF needed were 0.5 and 5.0 mM, respectively, and at least 60 min of preincubation was required.
Titration of Y331W F1 with MgADP in the Presence of
AlCl3 and NaF
Quenching of the fluorescence signal of Y331W mutant
F1 was used to determine nucleotide binding to catalytic
sites. It was found that inclusion of AlCl3 and NaF had no
effect on the fluorescence spectrum of the
Y331W enzyme. Initial
experiments were done on native enzyme, i.e. enzyme that had
been passed sequentially through two 1-ml Sephadex G-50 columns as
described under "Experimental Procedures." This treatment removes
catalytic site-bound nucleotide but not endogenous noncatalytic
site-bound nucleotide (27). For MgADP binding in the absence of
fluoroaluminate (Fig. 1,
circles), NaADP was added to enzyme in 50 mM
Tris/SO4, pH 8.0, in the presence of 2.5 mM
MgSO4, and the data were similar to what we have previously published (23, 24, 31). We previously determined that a model assuming
two types of binding site, with one site of higher and two sites of
lower affinity, gives a satisfactory fit to these data (23, 24, 31).
When the same model was used here, calculated values of binding
parameters were as follows: Kd1 = 0.08 µM, n1 = 1.21;
Kd2 = 14 µM, n2 = 1.62.
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For MgADP binding in the presence of fluoroaluminate (Fig. 1, triangles), NaADP was added to enzyme in 50 mM Tris/SO4, pH 8.0, in the presence of 2.5 mM MgSO4, 0.5 mM AlCl3, and 5 mM NaF. The effect of fluoroaluminate was very obvious, with a large increase in binding of MgADP occurring at low MgADP concentrations. It was found that a theoretical model assuming three binding sites of different affinities gave a better fit to these data than a model with two types of site. Calculated values for binding parameters were as follows: Kd1 < 1 nM, Kd2 = 0.06 µM, Kd3 = 40 µM. For comparison, if the data for MgADP binding in the absence of fluoroaluminate (Fig. 1, circles) were analyzed using the same three-site model, the calculated values for binding parameters were as follows: Kd1 = 0.05 µM, Kd2 = 2.54 µM, Kd3 = 47 µM. (The three-site model was equally satisfactory as the two-site model for these data.) Overall, the Fig. 1 binding curves indicated that fluoroaluminate induced a very large increase in affinity for MgADP at site one, a significant effect at site two, and little or no effect at site 3.
However, it should be noted that in the presence of fluoroaluminate (Fig. 1, triangles), at low concentrations of added NaADP, the stoichiometry of bound catalytic site MgADP considerably exceeded the actual amount of ADP added. The F1 concentration in Fig. 1 was 100 nM, and total MgADP concentration is plotted on the x axis. (It is assumed that total MgADP is equivalent to ADP added, since MgSO4 concentration was 2.5 mM). A possible explanation for the anomaly could be that endogenous ATP or ADP is released from noncatalytic sites and becomes sequestered into catalytic sites by fluoroaluminate, because the MgADP-fluoroaluminate is bound at catalytic site one with extremely high affinity.
This possibility was investigated as follows. Native enzyme was preincubated in the presence of 2.5 mM MgSO4, 0.5 mM AlCl3, and 5 mM NaF, and then ATPase activity was assayed and found to be inhibited by 60%. In a parallel fluorescence experiment, the addition of 2.5 mM MgSO4, 0.5 mM AlCl3, and 5 mM NaF to enzyme in 50 mM Tris/SO4, pH 8.0, induced a 10% quench of fluorescence. This fluorescence quench is equivalent to a catalytic site occupancy of 0.6 mol/mol, which is in excellent agreement with the degree of inhibition seen. In contrast, when nucleotide-depleted enzyme (depleted of catalytic and noncatalytic site nucleotide) was tested similarly, there was no inhibition of ATPase and only a small quench of fluorescence. Therefore, we concluded that in native enzyme, significant transfer of nucleotide from noncatalytic to catalytic sites did occur in presence of fluoroaluminate.
MgADP binding experiments were therefore repeated using nucleotide-depleted enzyme. For the data in the absence of fluoroaluminate (Fig. 2, circles), a model with two types of binding site gave a satisfactory fit to the data as seen previously (23, 24, 31), and the following values were calculated: Kd1 = 0.13 µM, n1 = 1.29; Kd2 = 6.8 µM, n2 = 1.37. These values are similar to our previously published values for nucleotide-depleted F1 and are similar to the values for native enzyme (see above). A model with three sites of differing affinities gave an equally satisfactory fit with calculated values of Kd1 = 0.07 µM, Kd2 = 1.1 µM, Kd3 = 14 µM.
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In the presence of fluoroaluminate (Fig. 2, triangles),
again a very large increase in binding stoichiometry at low MgADP concentrations was seen. These experiments utilized 26 nM
F1 concentration, which, under our experimental conditions,
is at the low range permissible for a satisfactory fluorescence signal.
In Fig. 2, we have plotted bound catalytic site MgADP versus
calculated free, nonbound MgADP concentration (x axis). Even
at the lowest concentration of free MgADP (1.7 nM), the
first catalytic site was already filled when fluoroaluminate was
present. A fit of the data to a model assuming two types of binding
site was unsatisfactory, but a model assuming three sites of different
affinities gave a satisfactory fit, and the calculated binding
parameters were as follows: Kd1 1 nM, Kd2 = 0.06 µM,
Kd3 = 5.6 µM. At concentrations of
MgADP below the lowest point shown in Fig. 2, calculated free MgADP
concentration was undependable, because it represented a tiny fraction
of the total MgADP present (bound plus free). The value for
Kd1 of
1 nM should be taken only
as indicating an extremely high affinity for MgADP-fluoroaluminate at
catalytic site one and not as an accurate estimate of binding affinity. A value far below 1 nM would not be surprising. Fig. 2
showed that Kd2 was also decreased in the presence
of fluoroaluminate as compared with its absence.
Summarizing the data of Figs. 1 and 2, fluoroaluminate had a very large effect on MgADP binding at catalytic site one, a more moderate effect at site two, and no significant effect at site three. The very tight MgADP-fluoroaluminate complex formed at catalytic site one may be regarded with confidence as a transition state analog, and it may well be that a complex with partial transition state-like properties forms also at catalytic site two. As noted under "Discussion," there is prior evidence for binding of 2 mol of MgADP-fluoroaluminate/mol of F1.
Titration of Y331W F1 with ADP in the Presence
of AlCl3 and NaF
Fig. 3 shows binding of ADP, in the
absence of Mg2+, to native Y331W mutant enzyme. It is
evident that fluoroaluminate did not influence ADP binding in the
absence of Mg2+. The data were analyzed assuming a model
with one class of binding site, as used previously for ADP binding (23,
24, 31), and gave values of Kd = 28 µM
and n = 2.8 in the absence or presence of
fluoroaluminate. Therefore, a transition state analog did not form
under these conditions. This is consistent with x-ray structures of
enzymes complexed with MgADP-fluoroaluminate, which show that
Mg2+ plays an important role in forming the complex itself
and in liganding the complex to the enzyme catalytic site. Inhibition of F1-ATPase by fluoroaluminate requires the presence of
Mg2+ (13), and the enzyme is inactive in the absence of
Mg2+.
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Taken together, the results of Table I and Figs. 1-3 demonstrate that
the fluorescence of residue -Trp-331 provides a sensitive probe of
high affinity binding of the MgADP-fluoroaluminate complex and of
formation of a catalytic transition state-like complex, at the first,
highest affinity catalytic site of F1-ATPase.
Effects of Mutations of Critical Catalytic Site Residues on Binding Affinity for the MgADP-fluoroaluminate Complex
Residue -Lys-155--
-Lys-155 is the Lys residue of the
Walker A consensus sequence. In the x-ray structure, it is seen to lie
close to the phosphate moieties of the catalytic site-bound nucleotide
(1), and as will be discussed later, it has previously been implicated
as a critical functional residue. Here our goal was to determine its
role in stabilizing the catalytic transition state using binding of
MgADP-fluoroaluminate as the assay.
Fig. 4A shows titration of the
native K155Q/
Y331W enzyme with MgADP in the presence and absence
of fluoroaluminate. The two curves are essentially identical, showing
there was no enhancement of MgADP binding by fluoroaluminate, in marked
contrast to the results seen with the parent
Y331W mutant
F1 in Fig. 1. This result demonstrates that formation of
the catalytic transition state does not occur in the
K155Q mutant,
giving direct confirmation of the role of
-Lys-155 in stabilizing
the transition state.
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Residue -Glu-181--
This residue was earlier implicated as a
critical catalytic residue by mutagenesis studies (see
"Discussion"). In the x-ray structure (1), residue
-Glu-181 is
seen to be located at some distance (4.1 Å) from the
-phosphate,
but it is close to a water molecule that appears hydrogen-bonded to the
carboxyl oxygens and could be the substrate water for hydrolysis. Our
goal was to analyze the role of
-Glu-181 in stabilizing the
transition state using the MgADP-fluoroaluminate binding assay.
Fig. 4B shows titration of the native E181Q/
Y331W
enzyme with MgADP in presence and absence of fluoroaluminate. There was no enhancement of MgADP binding by fluoroaluminate, demonstrating that
formation of the catalytic transition state does not occur in the
E181Q mutant.
Effects of DCCD Inhibition on Binding Affinity for the MgADP-fluoroaluminate Complex
DCCD reacts with E. coli F1 at residue
-Glu-192, the carboxyl oxygens of which are 16.6 Å away from the
-phosphate of catalytic site-bound MgAMPPNP in the x-ray structure
(1), too far removed for this residue to be involved directly in
transition state stabilization. Nevertheless, DCCD inhibits
steady-state ATPase potently. In a recent paper (32), we proposed a
mechanism for this inhibition (see "Discussion"), in which DCCD
does not inhibit the catalytic step per se. It was therefore
of interest to determine the effect of DCCD-reaction on formation of
the transition state. As we showed previously, the DCCD-inhibited
Y331W enzyme is readily amenable to assay of nucleotide binding by
fluorescence assay (27).
Native Y331W mutant F1 was reacted with DCCD as
described under "Experimental Procedures." MgATPase activity was
inhibited by 99%. MgADP binding to DCCD-inhibited enzyme was then
determined in the presence and absence of fluoroaluminate, as shown in
Fig. 5. In the absence of
fluoroaluminate, the data were similar to those obtained for the
uninhibited enzyme as in Fig. 1. A model with two classes of binding
site gave a satisfactory fit to the data, and the following
values were calculated: Kd1 = 0.02 µM, n1 = 0.6;
Kd2 = 4.2 µM,
n2 = 1.98. A model with three sites of different
affinities gave an equally satisfactory fit, and the calculated values
were as follows: Kd1 = 0.07 µM,
Kd2 = 3.7 µM, Kd3 = 11.3 µM. Therefore, reaction with DCCD had little
effect on MgADP binding affinity.
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However, in the presence of fluoroaluminate (Fig. 5,
triangles), it was apparent that MgADP binding affinity was
very greatly increased at low MgADP concentrations, just as for
uninhibited enzyme in Fig. 1. We again used a model assuming three
sites of different affinities to calculate binding parameters from
these data. The calculated values were as follows:
Kd1 < 1 nM; Kd2 = 0.15 µM; Kd3 = 29 µM.
The value for Kd1 should again not be taken as an
accurate assessment of binding affinity for MgADP-fluoroaluminate at
catalytic site one, since this site was already filled at the lowest
concentration of MgADP tested. The main point to be made is that the
results with DCCD-inhibited enzyme in Fig. 5 were essentially the same as those for uninhibited enzyme in Fig. 1. We conclude that in the
DCCD-inhibited enzyme a very large increase in binding affinity for
MgADP was induced by fluoroaluminate at catalytic site one and
therefore that DCCD reaction had little, if any, detrimental effect on
formation of the transition state.
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DISCUSSION |
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MgADP-fluoroaluminate complex is known to mimic the catalytic
transition state in numerous ATPase and GTPase enzymes (14-22). Here
we used binding of MgADP-fluoroaluminate to determine formation of the
transition state in F1-ATPase. Binding affinity for MgADP in the presence of fluoroaluminate at each of the three catalytic sites
in F1 was determined using the fluorescence signal of
residue -Trp-331, engineered specifically into the catalytic sites
in the
Y331W mutant (23, 24).
The results leave no doubt that MgADP-fluoroaluminate is a transition
state analog in F1-ATPase. Extensive biochemical data had
previously strongly indicated that this was the case (11-13). We found
that MgADP binding affinity at catalytic site one was very greatly
enhanced by fluoroaluminate and that Mg2+ was required for
this effect to occur. Furthermore, MgADP binding was not enhanced at
all by fluoroaluminate in mutant enzymes (K155Q and
E181Q) where
side-chains of critical catalytic site residues, located close to bound
nucleotide substrate, are modified. It should be noted that neither of
these residues plays any role in liganding the Mg2+ of the
magnesium-nucleotide substrate
(31).3 This new approach for
determination of the transition state is rapid, uses a low
concentration of enzyme in small volumes, and avoids the use of
radioactivity, features that make it attractive for future studies of
the transition state in normal, mutant, or inhibited enzyme.
As well as causing a very large increase in binding affinity for MgADP
at the first catalytic site, fluoroaluminate had a smaller effect on
MgADP binding at site two, indicating that an MgADP-fluoroaluminate
complex might also be bound to site two. In previous work, it was
demonstrated that the stoichiometry of binding of MgADP-fluoroaluminate
complex to mitochondrial F1 was 2 mol/mol (12, 13), based
on measurement of bound ADP, aluminum, and fluoride. Later work (33)
using kinetic methods with 3
3
subcomplex of thermophilic bacillus PS3 F1 has also
supported a total binding stoichiometry of 2 mol of
MgADP-fluoroaluminate/mol; however, this paper emphasized that full
inhibition of ATPase was already achieved at a binding stoichiometry of
1 mol/mol. Taken overall, we believe the data indicate that a true
transition state complex forms at catalytic site one, and a second
MgADP-fluoroaluminate complex, possibly with partial transition
state-like structure can also form at catalytic site two but not at
site three.
As we have pointed out in a recent review (5), it is an assumption common to all current proposals for the catalytic mechanism of F1-ATPase and ATP synthase that, under steady-state conditions at Vmax, the actual chemical bond cleavage and synthesis reaction of ATP hydrolysis or synthesis occurs in only one of the three catalytic sites at any one time. This work supports this assumption, if one accepts the above arguments that only one catalytic site forms a true transition state complex. Presumably, formation of the catalytic transition state conformation in any one catalytic site precludes its simultaneous formation in either of the other two sites. In this behavior, F1-ATPase is similar to the ABC transporter P-glycoprotein, where experiments using the transition state analog vanadate have demonstrated that, although both nucleotide-binding sites of P-glycoprotein are capable of catalysis, the transition state conformation can only be attained by one site at any one time (34).
In Fig. 4A, we show that fluoroaluminate did not enhance
MgADP binding in the K155Q/
Y331W mutant, indicating that the
catalytic transition state did not form. Residue
-Lys-155 is
established as critical for catalysis. Mutagenesis of this residue
impairs steady-state ATP hydrolysis rate by 3 or more orders of
magnitude (9, 35). Studies of the Glu and Gln mutant enzymes revealed that
-Lys-155 is functionally important for binding of MgATP, through the
-phosphate, particularly at catalytic sites one and two
(9, 10, 24). However, it plays no functional role in MgADP binding
(24). Unisite experiments (9, 10) indicated that it contributes to
stabilization of the catalytic transition state, in agreement with the
present data. We can therefore describe the role of this residue in
catalysis as follows. It provides binding energy in MgATP hydrolysis by
binding the MgATP and then stabilizes the transition state but not the
product MgADP. In the synthesis direction, movement of this residue
away from the
-phosphate of tightly bound MgATP, during the
rotation-induced "binding change," is critical to allow release of MgATP.
In Fig. 4B, we show that the E181Q/Y331W mutant
F1 also showed no formation of the catalytic transition
state complex. Mutagenesis of residue
-Glu-181 reduces the
steady-state ATP hydrolysis rate by several orders of magnitude,
depending on the mutation (9, 36, 37). In the Gln mutant, which reduces
hydrolysis by more than 3 orders of magnitude, it was found that
binding affinities of MgATP, MgADP, ATP, and ADP are all essentially
normal (24, 31), demonstrating that the effect on catalysis was not in
any way due to perturbation of binding of magnesium-nucleotide
substrate and that residue
-Glu-181 is not involved in
Mg2+ liganding. In agreement with the current work,
previous unisite experiments had indicated that the catalytic
transition state was destabilized in this mutant (9). A similar
conclusion can be drawn from the (less complete) set of unisite
parameters presented for the Ala mutant (36).
One can conclude, therefore, that residue -Glu-181 is uniquely
involved in generation of the catalytic transition state and that it
does not accelerate catalysis by providing nucleotide substrate binding
energy. In the x-ray structure,
-Glu-181 is located 4.1 Å from the
-phosphate of MgAMPPNP (1) and appears to be hydrogen-bonded to a
water molecule through its carboxyl oxygens. Its role in generating and
stabilizing the transition state is probably linked to its role in
immobilizing and polarizing the substrate water, therefore.
The experiments with DCCD provide additional information regarding the
mechanism of inhibition by this covalent inhibitor. Multisite MgATP
hydrolysis is inhibited potently by DCCD, but unisite catalysis is
inhibited only partly (38). At 99% inhibition of multisite catalysis,
as achieved in this work, the stoichiometry of DCCD reaction (with
residue -Glu-192) is 2 mol/mol of F1 (38). Tryptophan
fluorescence assays of MgATP binding to the DCCD-inhibited mutant
enzymes
Y331W and
F148W have recently been described and have led
to a description of the mechanism of inhibition by DCCD (32). Briefly,
it was concluded that DCCD acts by blocking a conformational signal
transmission event between catalytic sites that occurs upon binding of
MgATP to catalytic site three and that leads subsequently, in the
catalytic MgATPase mechanism of Weber and Senior (5), to rapid
hydrolysis of MgATP already bound at catalytic site one. Our
explanation of DCCD-inhibition proposes that the actual catalytic step
is not affected by DCCD reaction. The present experiments strongly
support our proposed mechanism for DCCD inhibition by demonstrating
that binding of MgADP-fluoroaluminate occurs normally in DCCD-reacted
enzyme. The ability of the enzyme to attain the catalytic transition
state is not affected by DCCD, although overall only 1% of activity remains. As noted above, DCCD reacts at a distance (16.6 Å) from the
catalytic site and could not interfere directly with transition state
formation or stabilization.
Abrahams et al. (1) in their analysis of the x-ray structure
of F1 suggested that reaction with DCCD could inhibit by
impeding conversion of the catalytic sites between conformational
states, similar to our proposal, but also they suggested that DCCD
might inhibit by impeding nucleotide access and binding to the
catalytic sites, due to introduction of the bulky cyclohexyl moieties
into a conical tunnel, which was proposed to allow nucleotide ingress and egress. While the results of this study would be in agreement with
the latter suggestion (and it is pertinent that DCCD does partly
inhibit unisite catalysis (38)), we feel that our previous data (32)
favor the inhibitory mechanism discussed here and in Ref. 32. The fact
that thermodynamic binding constants (Kd) are not
affected by DCCD modification (Ref. 27 and this paper) requires that
any change in association rate constants has to be accompanied by an
exactly parallel change in dissociation rate constants. In the
uninhibited enzyme, the distribution of nucleotides on the catalytic
sites under Vmax steady-state conditions (1 MgATP and 2 MgADP) establishes product MgADP release as the
rate-limiting step of the overall hydrolysis reaction (5, 32, 39). If the main effect of DCCD reaction were reduction of substrate binding and product release rates by the same factor, the product release step
would still be rate-limiting, and the MgATP/MgADP distribution should
remain the same. However, it is changed to 1.6:1.4 in the inhibited
enzyme (32), indicating that DCCD acts immediately subsequent to the
MgATP binding step. It further may be noted that mutagenesis of the
DCCD target residue -Glu-192 to Gln significantly inhibits multisite
ATP hydrolysis (by 93%; Ref. 9), and this occurs without introduction
of significant new bulk in this region of the protein.
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ACKNOWLEDGEMENT |
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We thank Rachel Shaner for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM25349 (to A. E. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
MgADP-fluoroaluminate complex is generated by
incubating together enzyme, MgADP, AlCl3, and NaF. The
fluoroaluminate species bound in the x-ray structures in different
enzymes (14-22) was either AlF3 or
AlF4. The fluoroaluminate species that
occurs at the catalytic site of F1 is not yet known from
x-ray data, and we will refer to it here simply as fluoroaluminate.
3
We did not examine the effect of mutation of any
of the known Mg2+-liganding residues of F1
(-Thr-156,
-Glu-185, or
-Asp-242; Ref. 31) in this work,
because, based on x-ray crystallography studies (14-22), it may be
anticipated that all will prevent binding of the MgADP-fluoroaluminate complex.
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ABBREVIATIONS |
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The abbreviations used are:
DCCD, dicyclohexylcarbodiimide;
AMPPNP, adenosine
5'-(,
-imino)triphosphate.
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REFERENCES |
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