(Received for publication, October 29, 1996, and in revised form, December 12, 1996)
From the Research Laboratory of Resources Utilization, Tokyo
Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan and the
Department of Chemistry and Biochemistry, University of
California at San Diego, La Jolla, California 92093-0601
A mutant
3
3
complex of
F1-ATPase from thermophilic Bacillus PS3 was
generated in which noncatalytic nucleotide binding sites lost their
ability to bind nucleotides. It hydrolyzed ATP at an initial rate with
cooperative kinetics (Km(1), 4 µM; Km(2), 135 µM) similar to the wild-type complex. However, the
initial rate decayed rapidly to an inactivated form. Since the
inactivated mutant complex contained 1.5 mol of ADP/mol of complex,
this inactivation seemed to be caused by entrapping inhibitory MgADP in
a catalytic site. Indeed, the mutant complex was nearly completely
inactivated by a 10 min prior incubation with equimolar MgADP. Analysis
of the progress of inactivation after initiation of ATP hydrolysis as a
function of ATP concentration indicated that the inactivation was
optimal at ATP concentrations in the range of
Km(1). In the presence of ATP, the
wild-type complex dissociated the inhibitory [3H]ADP
preloaded onto a catalytic site whereas the mutant complex did not.
Lauryl dimethylamineoxide promoted release of preloaded inhibitory
[3H]ADP in an ATP-dependent manner and partly
restored the activity of the inactivated mutant complex. Addition of
ATP promoted single-site hydrolysis of
2
,3
-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single
catalytic site of the mutant complex. These results indicate that
intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for
catalytic cooperativity of F1-ATPase observed at ATP
concentrations below ~300 µM.
F1-ATPase is the extrinsic membrane sector of
H+-ATP synthase and comprises five different subunits in a
stoichiometry of
3
3
1
1
1 (1). According to the crystal structure of bovine heart mitochondrial F1 (MF1)1 (2), the
and
subunits are arranged alternately to a form hexagonal
3
3. The six nucleotide binding sites are
located at different interfaces between the
and
subunits. The
three catalytic sites are mainly on the
subunits, whereas the three
other sites called noncatalytic nucleotide binding sites are mainly on
the
subunits. The overall structural topologies of the catalytic and noncatalytic sites are very similar to each other and both sites
contain the two sequences known as the Walker motif A and B, which are
commonly found in many nucleotide-binding proteins (3). Motif A, which
is also called as P-loop, has the consensus sequence,
GXXXXGK(T/S), and motif B consists of a stretch of four consecutive hydrophobic residues followed by Asp.
The function of the noncatalytic site is obscure. However, recent
studies suggest that the F1-ATPase is prone to develop
turnover-dependent inactivation and the noncatalytic sites
play a role in relieving the inactivation (4, 5). When
nucleotide-depleted MF1 or F1-ATPase from the
thermophilic Bacillus PS3 (TF1) hydrolyzes relatively low concentration of ATP, three kinetic phases are often
observed in the presence of an ATP regenerating system. An initial
burst rapidly decelerates to an intermediate rate that, in turn,
gradually accelerates to a final steady-state rate. It has been
postulated that transition from the initial phase to the intermediate
phase is caused by turnover-dependent entrapment of
inhibitory MgADP in a catalytic site (6-8), and transition from the
intermediate phase to the final phase reflects slow binding of ATP to
the noncatalytic sites, which promotes dissociation of inhibitory MgADP
from the affected catalytic site. After prior loading of a catalytic
site of MF1 (4, 9, 10), TF1 (5, 11), and
chloroplast F1-ATPase (12) with MgADP, the enzymes hydrolyze ATP with extended lag. The observation that the binding of
ATP to noncatalytic sites stimulates ATPase activity was also reported
(13-17). All these kinetic features are observed with the
3
3
complex of TF1 (18).
The
3
3
complex of TF1
containing
subunits with a mutation in the Walker motif B,
-D261N, dissociates inhibitory MgADP only slowly even in the
presence of ATP and the transition from the intermediate phase to the
final phase almost disappeared, exhibiting a low final rate of ATP
hydrolysis, about 30% of that of the wild-type complex (18).
Conversely, the
3
3
complex of
TF1 containing
subunits with a mutation in the Walker motif A,
-T165S, efficiently dissociates inhibitory MgADP and exhibits a severalfold higher final rate of ATP hydrolysis than that of
the wild-type complex (19). These results suggest that F1-ATPase in the inactivated state with inhibitory MgADP in
a catalytic site is reactivated by ATP binding to noncatalytic sites. However, important unanswered questions remain. For instance, does
enzyme containing inhibitory MgADP in a single catalytic site have weak
residual ATPase activity or is it completely inactive? Is release of
inhibitory MgADP totally dependent on ATP binding to noncatalytic sites
or is there slow release of inhibitory MgADP that is independent of ATP
binding to noncatalytic sites?
The role of noncatalytic site in the cooperative kinetics of
F1-ATPase also remains to be clarified.
F1-ATPase exhibits negative cooperativity characterized
with two or three apparent Km values which are 1-30
µM, 100-300 µM, and above 400 µM (20-24). This apparent negative cooperativity is
observed also for the membrane-bound enzyme (25) and proton
translocation (26). Slow binding of ATP to noncatalytic sites can
explain apparent negative cooperativity at relatively high
concentration of ATP represented by the highest Km
value (4). Weber et al. reported a single
Km value for the mutant F1-ATPase from
Escherichia coli (EF1) with mutations
-D261N/
-R365W in which nucleotide binding to noncatalytic sites
was greatly diminished (27). They stated that the kinetics of this
mutant showed no deviation from simple monophasic Michaelis-Menten
kinetics. However, they assayed ATPase activity by measuring
Pi release, which is not suitable to monitor fluctuation in
rate during assay. Therefore, as stated in their paper, they did not
scrutinize the kinetic behavior at very low substrate concentrations,
which is necessary to detect a Km at 1-30
µM. Similarly, Yohda et al. reported that
(D261N)3
3
complex of TF1
did not exhibit cooperativity, but again they examined kinetics only
above 20 µM ATP (28). Therefore, the effect of
noncatalytic sites on cooperative kinetics of F1-ATPase
remains unsettled.
Since the covalent modification of the noncatalytic sites with
5-p-fluorosulfonylbenzoyladenosine inactivates ATPase
activity completely (29), it is even possible to argue that
noncatalytic sites are essential for the activity of
F1-ATPase, although their participation in catalysis is
indirect. The mutants reported so far whose noncatalytic sites are
impaired, namely EF1(
-D261N/
-R365W) and
(D261N)3
3
complex of TF1,
have considerable ATPase activity (18, 27, 28). Especially, the
EF1 mutant showed ATPase activity even under the condition
where noncatalytic sites were supposed to be empty and Weber et
al. concluded that occupancy of the noncatalytic sites by adenine
nucleotides was not required for catalysis. However, ambiguity remains
because both EF1(
-D261N/
-R365W) and
(D261N)3
3
complex of TF1
have the ability to bind nucleotide to the noncatalytic sites even
though the affinity is decreased.
To obtain more discriminating data on the role of noncatalytic sites,
it is necessary to characterize a mutant F1-ATPase that completely lacks the ability to bind nucleotides to noncatalytic sites.
To generate such a mutant, we have replaced four amino acid residues in
Walker motif A and B sequences of the TF1- subunit by
Ala residues, and analyzed nucleotide binding properties and ATP
hydrolysis catalyzed by the
3
3
complex
containing the mutated
subunits under a wide range of ATP
concentration. Comparison of this mutant
3
3
(
NC) complex and the wild-type
3
3
complex has revealed the essential
role of noncatalytic sites in steady-state catalytic turnover of
F1-ATPases.
E. coli strains
used were JM109 (30) for preparation of plasmids, CJ236 (31) for
generating uracil-containing single-stranded plasmids for site-directed
mutagenesis, and JM103(uncB-uncD) (32) for expression of the
wild-type and
NC
3
3
complexes of
TF1. Plasmids pTABG1 and pKABG1 (33), which carried genes for the
,
, and
subunits of TF1, were used for
mutagenesis and for gene expression, respectively. Terrific broth (34)
was used as a culture media and supplemented with ampicillin (50~100 µg/ml). Helper phage M13K07 was obtained from Pharmacia Japan, Tokyo.
The expression plasmid for the
NC complex, the noncatalytic sites of
which are incapable of binding of adenine nucleotides, was constructed
as follows. The four mutations,
-Lys-175
Ala,
-Thr-176
Ala,
-Asp-261
Ala, and
-Asp-262
Ala, were introduced into
pTABG1 by using two synthetic oligonucleotides;
5
-AATGGCGACGGCCCCGTTTGT-3
and
5
-TGCTTCGATACCGATCACAAC-3
(changed
bases are underlined) (31). The EcoRI-BglII
fragment from the resultant plasmid was ligated into the
EcoRI-BglII site of pKABG1 to produce the
expression plasmid, pKABG1-
K175A/
T176A/
D261A/
D262A.
Recombinant DNA procedures were performed as described in the manual
(34).
2-N3-[3H]ATP and
2-N3-[3H]ADP were synthesized and purified as
described elsewhere (35). [3H]ADP was purchased from
DuPont NEN. Lauryldimethylamineoxide (LDAO) was purchased from
Calbiochem. Synthesis and purification of
2,3
-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) were carried
out as described previously (36). The wild-type and
NC
3
3
complexes were purified as
described before (33). Purified complexes did not contain detectable
amount of endogenously bound adenine nucleotide (<0.05 mol/mol of
complex).
Protein concentrations of
TF1 and 3
3
complexes were
determined by measurement of absorbance at 280 nm using the factor 0.45 of absorbance for 1 mg/ml of protein. ATPase activity was measured at
25 °C in the presence of an ATP regenerating system. An assay
mixture contained 50 mM Tris-Cl (pH 8.0), 100 mM KCl, indicated concentrations of ATP, 2.5 mM
phosphoenolpyruvate, 50 µg/ml pyruvate kinase (rabbit muscle), 50 µg/ml lactate dehydrogenase (pig muscle), and 0.2 mM
NADH. Pyruvate kinase and lactate dehydrogenase (Boehringer Japan,
Tokyo) were diluted from solution in glycerol. These amounts of
auxiliary enzymes were confirmed to be sufficient for the rapid ATP
hydrolysis in the initial burst phase of catalysis. MgCl2
concentration was maintained at 2 mM excess over that of ATP in the assay mixture. Typically, the reaction was initiated by
addition of the enzyme to 2 ml of the assay mixture and the rate of ATP
hydrolysis was monitored as the rate of oxidation of NADH determined by
the absorbance decrease at 340 nm. The data were stored in an on-line
computer for further analyses. We attached a device on the photometer
lid that enabled us to start the reaction by injecting the enzyme
solution without opening it. The spectrophotometer was equipped with a
small stirrer to ensure rapid mixing. We confirmed that the maximum
dead time of measurement was below 4 s after the start of the
reaction and the data from 5 s to 20 s were usually used for
analysis. The initial rates were obtained from exponential extrapolation of the experimental data between 5 and 20 s to time zero. One unit of activity was defined as the activity that hydrolyzed 1 µmol of ATP/min. Assessment of nucleotide binding to catalytic and
noncatalytic sites of the complexes by photoaffinity labeling with
2-N3-[3H]AT(D)P was carried out as described
previously (37). Briefly, the solution (100 µl) containing 1.5 mg of
the wild-type or
NC
3
3
complex, 150 µM 2-N3-[3H]AT(D)P (2700 cpm/nmol), 2 mM MgCl2, 100 µM
EDTA, and 50 mM Tris-Cl (pH 7.5), was irradiated for 40 min
at room temperature with a Minerallight, and digested by
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated-trypsin after denaturation of the proteins and removal
of unbound nucleotides. An aliquot of the digested solution was
injected into a C4 reversed-phase HPLC column and developed with a
gradient of CH3CN in 0.1% HCl as follows: 0-10 min, 0%; 10-100 min, 0-24%; 100-115 min, 24-48%, 115-120 min, 48-80%.
Fractions, 1 ml each, were collected and radioactivity of each fraction
was measured. Difference spectra induced by the interaction between TNP-ATP and the proteins and single-site hydrolysis of TNP-ATP were
measured according to previous papers (38-40). The bound nucleotide content of the enzyme complex was determined after separating free
nucleotide from enzyme-bound nucleotides by centrifuge elution using a
1-ml column of Sephadex G-50, extracted with perchloric acid, and
analyzed by HPLC (38). Release of [3H]ADP from the
complexes was monitored according to the methods described by Jault
et al. (18).
The crystal structure of MF1 shows that, in
the noncatalytic nucleotide binding site, Lys and Thr in the Walker
motif A and Asp in the motif B of the subunit lie close to the
terminal phosphate and Mg2+ of bound Mg-AMP-P(NH)P, a
substrate analogue (2). In addition, the conserved Asp just adjacent to
the Asp of the motif B sequence of the
subunit also contributes to
the noncatalytic nucleotide binding site. The residues of
TF1-
subunit equivalent to the above residues of
MF1-
subunit are
-Lys-175,
-Thr-176,
-Asp-261, and
-Asp-262. Therefore, we replaced these residues of
TF1-
subunit by Ala residues. Four mutations,
-Lys-175
Ala,
-Thr-176
Ala,
-Asp-261
Ala, and
-Asp-262
Ala, were simultaneously introduced into the
subunit gene on an expression plasmid for
3
3
complex. Although it was reported
that the mutations at
-Lys-175 impaired assembly of subunit
complexes (27, 28, 41), the mutant
subunit constructed here
assembled normally into
3
3
complex
with the
and
subunits. The
NC complex was stable and
purified to homogeneity by the same method used for the purification of
the wild-type complex including the incubation at 60 °C for 30 min.
To assess binding of
adenine nucleotides to the noncatalytic sites, tryptic digests from the
wild-type and NC
3
3
complexes which
were photolabeled with 2-N3-[3H]ATP in the
presence of Mg2+ were analyzed. The profiles of tryptic
peptides resolved by reversed-phase HPLC are shown in Fig.
1. Elutions were carried out under the same conditions
reported previously in which assignment of radioactive peaks was
established (42). A radioactive peak eluted at around 78 min contains
the tryptic peptide with
-Tyr-364 derivatized, which is a part of
the noncatalytic site, and peaks eluted between 90 and 100 min contain
the tryptic peptides with
-Tyr-341 derivatized, which is a part of
the catalytic site (43). It has been shown that the tryptic peptides
derived from the catalytic site are often eluted as two (or more) peaks
as shown in Fig. 1A because of the heterogeneity arising
from hydrolysis of ATP tethered to
-Tyr-341 (35, 37). When the
elution profile of the
NC complex (Fig. 1B) is compared
with that of the wild-type
3
3
complex (Fig. 1A), it is obvious that the former does not have a
peak at around 78 min (shown by an arrow). The experiments
with 2-N3-[3H]ATP in the absence of
Mg2+, 2-N3-[3H]ADP in the absence
and presence of Mg2+ gave the same results; there was no
radioactive peak at the position corresponding to a peptide derived
from noncatalytic sites whereas a peak corresponding to a peptide from
catalytic site was always detected (data not shown). These results show
that 2-N3-adenine nucleotides cannot bind to the
noncatalytic sites of the
NC complex.
The binding of nucleotide was further examined by TNP-ATP-induced
difference spectra (Fig. 2). The difference absorption
spectra induced by binding of TNP-AT(D)P to the isolated wild-type or
subunit are significantly different from each other. A trough at
450 nm and a peak at 510 nm were observed for the
subunit, whereas
a trough at 395 nm and a peak at around 420 nm were observed for the
subunit (Fig. 2, uppermost and lowermost
traces) (38). Therefore, it is possible to determine the subunit
localization of the TNP-AT(D)P binding site of the complex by this
means. It has been shown that the wild-type
3
3
complex binds TNP-ADP preferentially to a single high affinity catalytic site on the
subunit until molar ratio of TNP-ADP to F1-ATPase is
1.25:1. After this site is filled, the second site occupied by TNP-ADP is a noncatalytic binding site on the
subunit (40). Similar binding
characteristics were observed for TNP-ATP and, for example, a large
contribution by the
subunit specific difference spectrum is obvious
in the difference spectrum at a 4:1 TNP-ATP·wild-type
3
3
complex molar ratio (Fig. 2). In
contrast, the
NC complex showed spectra typical for the
subunit
even at a 6:1 TNP-ATP·complex molar ratio (Fig. 2). This indicates
that TNP-ATP binds exclusively to the catalytic sites on the
subunits of the
NC complex and that noncatalytic sites on the
subunit is unable to bind TNP-ATP. Based on these results, we conclude
that the noncatalytic sites of the
NC complex do not bind adenine
nucleotides.
ATPase Activity of the
Fig. 3 shows time courses of ATP
hydrolysis by the wild-type and NC
3
3
complexes in the presence of an ATP
regenerating system. The wild-type complex hydrolyzed 20 µM ATP in three phases (trace b), an initial
burst decelerated to an intermediate phase that then accelerated to a
final state (18). At 2 mM ATP, the transition from the
intermediate phase to the final phase was not seen and it appeared to
proceed in two phases, an initial burst phase and a following
decelerated constant phase (trace d). The
NC complex also
hydrolyzed 20 µM and 2 mM ATP with an initial
burst (traces f and i). The rates of the initial
bursts by
NC complex at both concentrations were very similar to
those observed for the wild-type complex which are shown by overlaid dotted lines (traces g and j). However, the
initial burst of the
NC complex rapidly decelerated and hydrolysis
stopped in a short period. We analyzed the kinetics of initial rates of
the burst phase of the
NC complex at a wide range of ATP
concentrations (1-2000 µM) and compared them to those of
the wild-type complex (Fig. 4). Although there were some
differences at high ATP concentrations, the
NC complex hydrolyzed
ATP in a similar manner to the wild-type complex. As shown in the
inset of Fig. 4, the Eadie-Hofstee plots of the initial
rates of ATP hydrolysis by the wild-type and
NC complexes are
concave downward, indicating that ATP hydrolysis by the
NC complex,
like the wild-type complex, exhibits negative cooperativity. Apparent
kinetic parameters are calculated by a non-linear regression
curve-fitting (24) and summarized in Table I. At least
two sets of Km and Vmax are
necessary to simulate the experimental data for both wild-type and
NC complexes. The values of Km(1) and
Vmax(1) obtained for the
NC complex are
almost the same as Km(1) and
Vmax(1) of the wild-type complex.
Km(2) and Vmax(2)
of the
NC complex are also close (about 70%) to those of the
wild-type complex. Thus, the
3
3
complex without functional noncatalytic sites exhibits cooperative
kinetics which are very similar to those of the
3
3
complex with intact noncatalytic
sites.
|
Although the initial rate of ATP hydrolysis by the NC
complex obeys similar kinetics to those of the wild-type complex, it is
rapidly inactivated during catalytic turnover and hydrolysis stops
completely in a short period as described above (Fig. 3, traces
f and i). The dependence of the rate of inactivation on ATP concentration is shown in Fig. 5 (A and
B). The time course of inactivation was exponential curve,
and the first order rate constants of inactivation were obtained at
various ATP concentrations. As shown in Fig. 5C, the rate
constants of inactivation exhibit monophasic dependence on ATP
concentration. The ATP concentration that gave a half-maximal rate of
inactivation (apparent Kd) was 5 µM.
This value agrees well with the Km(1) (4 µM) obtained from analysis of the rate in the initial
burst phase. The maximal rate of inactivation was 0.33 s
1. Combining the rate of inactivation and the rate of
ATP hydrolysis, we can calculate the average number of catalytic
turnovers required for the inactivation at each ATP concentration. For
example, at 0.53, 1.1, 4.4, 11, 22, and 33 µM ATP, 17, 31, 63, 80, 97, and 105 turnovers are required for the inactivation,
respectively. Therefore, inactivation is not simply proportional to
turnover number. When ATP concentration is low, the enzyme is
inactivated in relatively few turnovers. More turnovers are required
for inactivation as ATP concentrations increased. Accordingly, the size
of initial burst is smaller when ATP concentration is low than when it
is high as seen in Fig. 5 (A and B).
Entrapment of Inhibitory MgADP in a Catalytic Site
To analyze
bound nucleotides of the inactivated NC complex, the
NC complex
was inactivated by a 20-min incubation with 20 µM ATP in
the presence of 2 mM MgCl2 and free nucleotides
were removed by passing through the Sephadex G-50 centrifuge column. The complex recovered in the effluent did not have ATPase activity, ensuring that it was still in an inactivated state. Analysis of the
bound nucleotides revealed that the inactivated
NC complex contained
1.5 mol of ADP/mol of the complex, while a control
NC complex that
was not previously exposed to nucleotide contained none (<0.05
mol/mol). The role of the bound ADP in the inactivated complex was
further investigated after incubating the complex with MgADP. As
reported previously (5), the wild-type complex with preloaded MgADP by
a prior incubation with equimolar MgADP for 5 min showed attenuated
initial rates of hydrolysis of 20 µM and 2 mM
ATP rather than initial burst, that accelerated gradually to a constant
phase (Fig. 3, traces a and c). In contrast, the
NC complex with preloaded MgADP could not hydrolyze ATP at all (Fig.
3, traces e and h). Reactivation did not occur
even after long incubation (5 h) of the assay mixture. To determine the
stoichiometry of inhibitory MgADP bound, the
NC complex was
preincubated with MgADP at various molar ratios to the complex for 10 min, and the residual ATPase activities at the initial burst phase were
measured in the presence of 2 mM ATP. As shown in Fig.
6, the extent of inactivation of the
NC complex was
almost proportional to the amount of added MgADP until the
concentration of MgADP reached to a 1:1 molar ratio to the complex
where nearly complete inactivation was observed. Since noncatalytic
sites in the
NC complex cannot bind MgADP, we conclude that the
NC complex is completely inactivated by entrapping MgADP in a single
catalytic site.
Effect of LDAO
It has been shown in previous studies that the
ATPase activities of TF1 and the wild-type
3
3
complex are stimulated
significantly by the neutral detergent LDAO (5, 18, 44). The
stimulation of ATPase activity by LDAO is thought to promote release of
inhibitory MgADP from a catalytic site in the presence of ATP (19).
When LDAO was present in the reaction mixture, the wild-type complex hydrolyzed 2 mM ATP at a constant rate from the beginning.
The constant rate in the presence of LDAO was very close to the rate in
the initial burst in the absence of LDAO (Fig.
7A). It appears that LDAO does not affect the
rate of ATP hydrolysis in the initial burst, but rather allows the
initial burst to continue linearly without deceleration. Similar to the
wild-type complex, LDAO had little effect on the initial burst of the
NC complex (Fig. 7B). However, different from the
wild-type complex, the initial burst decelerated even in the presence
of LDAO and reached a slow, final rate. When LDAO was added to the
NC complex, which was previously inactivated by aging in the assay
mixture or by prior incubation with MgADP, the same slow rate was
restored (Fig. 7C). The above experiments were performed at
2 mM ATP. However, a similar effect of LDAO on the
NC
complex was observed when it hydrolyzed 20 µM ATP (data
not shown). The stimulating effect of LDAO on the final steady-state
hydrolysis was saturated at 0.04% LDAO for the wild-type complex and
was nearly (but not completely) saturated at 0.15% LDAO for the
NC
complex (Fig. 7D). If the mechanism of action of LDAO is
only to amplify the conformational signal generated by the ATP binding
to noncatalytic sites that causes release of inhibitory MgADP, then
LDAO would not have an effect on the
NC complex. Probably, LDAO has
a direct effect on the catalytic site occupied by inhibitory ADP. This
effect might be small because the extent of activity restored by LDAO
in the case of the
NC complex is much smaller than that of the
wild-type complex as described above.
Release of Inhibitory MgADP
The effect of ATP and LDAO on the
release of preloaded, inhibitory [3H]ADP from the
wild-type and NC complexes was examined (Fig. 8). For
the wild-type complex, release of preloaded [3H]ADP was
promoted by ATP but not by LDAO (Fig. 8A). However, when
both ATP and LDAO were present in the solution, the release was greatly
enhanced and most [3H]ADP was released in 30 s. For
the
NC complex, neither ATP nor LDAO, when added alone, promoted
release of [3H]ADP. A moderate promotion of the release
was observed only in the presence of both ATP and LDAO (Fig.
8B). Based on these results, an explanation can be given for
the observations shown in Figs. 3 and 7. For the wild-type complex,
both entrapping and release of inhibitory MgADP occur during turnover
in the presence of ATP. A fraction of the complex is always in an
inactive state during steady-state catalysis in the absence of LDAO.
However, LDAO promotes release of the inhibitory MgADP from the
affected catalytic site and this converts nearly all of the enzyme to
an active state. This equilibrium was driven in the direction of
release of inhibitory MgADP when LDAO is present in the assay mixture
and, as a consequence, almost all of the enzyme is in an active state,
showing uninhibited ATPase activity. For the
NC complex, in
contrast, the presence of ATP does not promote release of inhibitory
MgADP. Once inhibitory MgADP is entrapped, it fails to dissociate
keeping the complex in an inactivated state. When LDAO and ATP are
present, inhibitory MgADP is released slowly and an equilibrium is
established with a small fraction of the complex free of inhibitory
MgADP resulting in partial restoration of activity.
Single-site TNP-ATP Hydrolysis and Chase-promotion
TF1 (38) and the
3
3
complex (40) hydrolyze TNP-ATP
slowly when TNP-ATP is added to the enzyme in a substoichiometric molar
ratio. Slow hydrolysis of TNP-ATP is greatly accelerated by
chase-promotion with ATP. It has been suggested that the ATP binding
site responsible for chase-promotion is the second catalytic site to be
filled (39, 45). Fig. 9 illustrates time courses of
TNP-ATP hydrolysis by the wild-type and
NC complexes. Similar to the
wild-type complex, the
NC complex slowly hydrolyzed
substoichiometric TNP-ATP and chase-promotion with ATP accelerated the
hydrolysis of the TNP-ATP. From this result we conclude that
participation of noncatalytic sites is not necessary for cooperativity
between two catalytic sites.
This work provides solid support for the view that
entrapping inhibitory MgADP at a catalytic site, either during
incubation with MgADP or during turnover under assay conditions, causes
inactivation of F1-ATPase. In addition, it is now clear
that enzyme that retains inhibitory MgADP at a single catalytic site is
completely inactive in ATP hydrolysis (Figs. 3 and 6). Differing from
the wild-type enzyme, inhibitory MgADP is not released from the NC
complex even in the presence of ATP (Fig. 8). Since catalytic sites in the
NC complex are as intact and available for ATP binding as those
of the wild-type complex, the failure of ATP to promote release of
inhibitory MgADP from a catalytic site can only be attributed to the
lack of ability of noncatalytic sites to bind ATP. Thus, it is
concluded that ATP binding to noncatalytic sites is essential for
continuous catalytic turnover. This may have physiological importance
since Richard et al. suggested that ATP synthesis by
H+-ATP synthase from thermophilic Bacillus PS3
was stimulated when the noncatalytic sites were occupied by ATP
(46).
Comparison of the rates of ATP
hydrolysis in the initial burst by the wild-type and NC complexes
revealed that both enzymes obey very similar cooperative kinetics (Fig.
4, Table I). Both the Km value and the
Vmax value of the
NC complex are almost the
same or close to the corresponding values of the wild-type complex. In
addition, the
NC complex can catalyze single-site hydrolysis and
chase-promotion using TNP-ATP as a substrate (Fig. 9). The remarkably
similar kinetics of the
NC complex and the wild-type complex
strongly indicates that the catalytic sites of the
NC complex are
intact and behave in a similar manner to the wild-type complex. In
other words, noncatalytic sites contribute little, if anything, to the
cooperative kinetics of the
3
3
complex. Thus, cooperative features of ATP hydrolysis by
F1-ATPase characterized with two sets of parameters,
Km(1) = 1-30 µM and
Km(2) = 100-300 µM,
reflect catalytic site to catalytic site cooperativity. The apparent
Km(2) of 140 µM observed
here agrees well with the Km for proton
translocation which was membrane potential independent (47).
Cooperativity observed at high ATP concentration (>400
µM) at steady-state catalysis is a phenomenon attributed
to slow nucleotide binding to noncatalytic sites (4).
Interestingly, the rate
of the progressive inactivation of the NC complex during turnover
shows a hyperbolic dependence on ATP concentration and exhibits an
apparent Kd of 5 µM (Fig. 5). This
Kd value corresponds to
Km(1) (4 µM) obtained from
initial rate analysis. This Km is thought to reflect
a catalytic cycle operating when two catalytic sites are occupied,
so-called bi-site catalysis (22). Owing to the very low
Kd and kcat values for ATP hydrolysis when only one catalytic site is occupied, single site catalysis is not
amenable to steady-state kinetic analysis. This means that occupancy of
two catalytic sites promotes transition from an active to an inactive
enzyme. On the other hand, we observed that loading a catalytic site
with exogenous MgADP is sufficient to inactivate the
NC complex
completely (Fig. 6). This apparent contradiction can be accommodated as
follows. The formation of inactivated MgADP·TF1 from
TF1 and MgADP is a slow process (2 M
1 s
1) (11) and this is also
the case for the
3
3
complex. The rate-limiting step is most likely to be the isomerization from a
transient, active MgADP·enzyme complex into the stable, inactive MgADP·enzyme complex (6, 19). If ATP hydrolysis operating with two
catalytic sites can facilitate the isomerization, ATP hydrolysis
characterized by Km(1) becomes an
apparently responsible step for the generation of the inactive complex.
After isomerization, MgADP at one of the two catalytic sites might be released during gel filtration procedures to analyze bound nucleotide. In this mechanism, cooperative interaction between two catalytic sites
is assumed to accelerate not only catalysis but also generation of
inactive species of the enzyme. It is interesting to note that the
inhibition of F1-ATPase by azide also progresses during
turnover and the rate of progress is dependent on the ATP binding
(and/or hydrolysis) characterized by the Km of about
10 µM (48), a value similar to the apparent
Kd above mentioned. Evidence suggests that azide
stabilizes the inhibitory MgADP·F1-ATPase complex (7,
19). Azide inhibition and inactivation of the
NC complex during
turnover probably operate by a similar mechanism.