The presence of phosphate at a catalytic site suppressesNoriyo Mitome, Sakurako Ono, Toshiharu Suzuki, Katsuya Shimabukuro, Eiro Muneyuki and Masasuke Yoshida Chemical Resources Laborato
Trang 1The presence of phosphate at a catalytic site suppresses
Noriyo Mitome, Sakurako Ono, Toshiharu Suzuki, Katsuya Shimabukuro, Eiro Muneyuki
and Masasuke Yoshida
Chemical Resources Laboratory, Tokyo Institute of Technology, Japan
F1-ATPase is inactivated by entrapment of MgADP in
catalytic sites and reactivated by MgATP or Pi Here, using a
mutant a3b3c complex of thermophilic F1-ATPase
(aW463F/bY341W) and monitoring nucleotide binding by
¯uorescence quenching of an introduced tryptophan, we
found that Pi interfered with the binding of MgATP to
F1-ATPase, but binding of MgADP was interfered with to a
lesser extent Hydrolysis of MgATP by F1-ATPase during
the experiments did not obscure the interpretation because
another mutant, which was able to bind nucleotide but not
hydrolyse ATP (aW463F/bE190Q/bY341W), also gave the same results The half-maximal concentrations of Pi that suppressed the MgADP-inhibited form and interfered with MgATP binding were both 20 mM It is likely that the presence of Piat a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme±MgADP±Pi complex, an active intermediate in the catalytic cycle Keywords: competition; FoF1-ATP synthase; MgADP inhibition; nucleotide binding; tryptophan ¯uorescence
FoF1-ATP synthase synthesizes ATP from ADP and
inorganic phosphate (Pi) by using the energy of proton
¯ow driven by a transmembrane electrochemical proton
gradient [1±3] The enzyme consists of a cytoplasmic
domain, referred to as F1-ATPase or F1, which carries
catalytic sites for the synthesis and hydrolysis of ATP, and a
membrane integral domain, Fo, which conducts protons
across the membrane F1has a subunit composition a3b3cde
and can be reversibly separated from Fo There are six
nucleotide-binding sites in F1-ATPase, three of which are
catalytic sites, located on the b subunits, and three other
noncatalytic sites, located on the a subunits In the crystal
structure of bovine mitochondrial F1-ATPase, the three a
and three b subunits are arranged alternately like segments
of an orange around the central coiled-coil structure of the
c subunit [4]
During ATP hydrolysis, FoF1-ATP synthase tends to
entrap MgADP, resulting in the generation of the so-called
MgADP-inhibited form [5±9] It has been shown that
MgADP inhibition is induced by occupation of a single
catalytic site by MgADP [10] During catalysis,
simulta-neous occupation of two catalytic sites promotes the onset
of this inhibition [11] The crystal structure of mitochondrial
F1-ATPase obtained in the presence of ADP, AMPPNP,
and NaN3 in 1994 [4] probably represents this
MgADP-inhibited form The MgADP-MgADP-inhibited form is activated by
ATP binding to the noncatalytic sites [12±15] An apparent
Kd for the activation process was deduced to be 430 lM using nucleotide-depleted mitochondrial F1-ATPase [14] Previously, we have reported that the mutant a3b3c subcomplex (DNC), which is defective in the noncatalytic nucleotide-binding site, is unable to continue ATP hydro-lysis because all the subcomplexes remain in the MgADP-inhibited form [16] However, the ATPase-inactive DNC
FoF1-ATP synthase catalysed continuous turnover of ATP synthesis [17] To explain this, it was proposed that the MgADP-inhibited form was not generated in the ATP synthesis reaction Furthermore the DNC a3b3c subcomplex was activated by Piand showed continuous ATP hydrolysis activity in the presence of Pi [18] Pi also activated the MgADP-inhibited form of the wild-type a3b3c subcomplex [18,19], F1[5,9] and FoF1-ATPase [11, 20, 21] in a similar manner These studies suggested that Pibound to catalytic site(s) and suppressed formation of the MgADP-inhibited form
In this study, we tried to elucidate the mechanism by which Piprevents the formation of the MgADP-inhibited form and the relationship between Pi binding and nucleotide binding at catalytic sites For this purpose,
we used the a(W463F)3b(Y341W)3c mutant subcomplex in which a Tyr residue near the catalytic site (Y341) was replaced by Trp to monitor nucleotide binding to the catalytic sites In addition, aW463 was replaced by Phe
to reduce the background ¯uorescence The ¯uorescence decrease of the introduced tryptophans near the catalytic sites re¯ects nucleotide binding to catalytic sites [22±24]
It was found that the presence of Pi at a catalytic site prevents the formation of the MgADP-inhibited form even if MgADP is still bound at that catalytic site The results suggest a mechanism of reactivation of the MgADP-inhibited form by Pi, in which Pi at a catalytic site shifts the equilibrium from the MgADP-inhibited form to an enzyme±MgADP±Pi complex, which is an active intermediate in the catalytic cycle
Correspondence to E Muneyuki, Chemical Resources Laboratory,
Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503,
Japan Fax: + 81 45 924 5277, Tel.: + 81 45 924 5232,
E-mail: emuneyuk@res.titech.ac.jp
Enzymes: F o F 1 -ATPase (EC 3.6.6.14); pyruvate kinase (EC 2.7.1.40);
lactate dehydrogenase (EC 1.1.1.27).
(Received 23 July 2001, revised 25 September 2001, accepted 22
October 2001)
Trang 2M A T E R I A L S A N D M E T H O D S
Strains, plasmids, and proteins
Escherichia coli strain JM109 was used [25] for preparation
of the plasmids pKABG1-aW463F was prepared as
previously described [26] The mutation of bY341W was
introduced into pTABG1 as previously described [23] The
MulI±SmaI fragment of pTABG1, which contained the
bY341W mutation, was ligated into the corresponding site
of pKABG1-aW463F to produce the aW463F/bY341W
mutant The MluI±BstPI fragment of pUCb-E190Q [27],
which contained the bE190Q mutation, was ligated into the
corresponding site of pKABG1-aW463F/bY341W to
pro-duce pKABG1-aW463F/bE190Q/bY341W
The subcomplexes were expressed and puri®ed as
described previously [26] Before use, the enzymes, which
were stored as ammonium sulfate suspensions, were puri®ed
on a Superdex 200 gel-®ltration column (Pharmacia
Bio-tech) with 100 mM potassium phosphate buffer (pH 7.0)
containing 1 mM EDTA The puri®ed fraction was
adsorbed on a Butyl-Toyopearl column (Tosoh), which
was equilibrated with 100 mM sodium phosphate buffer
(pH 7.0) containing 50 mM Mops/KOH, 50 mM KCl,
1 mMEDTA, and 20% saturated ammonium sulfate The
column was washed with the same buffer to deplete it of
nucleotide, and the enzyme was eluted with 50 mMMops/
KOH buffer (pH 7.0) containing 1 mMEDTA The eluted
enzyme was passed through a gel-®ltration column PD10
(Pharmacia Biotech) pre-equilibrated with 50 mM Mops/
KOH buffer (pH 7.0) containing 50 mM KCl and 2 mM
MgCl2 and used for the following experiments The
preparation contained less than 0.05 mol adenine nucleotide
per mol enzyme
The ATPase activity of the enzyme preparation at 4 mM
MgATP depended on the KCl concentration in the assay
mixture At 50 mM KCl, the activity was 12±13 Uámg)1,
whereas at 160 mMKCl, it was 30±40 Uámg)1, which are
in the normal range for thermophilic F1-ATPase activity
ATP hydrolysis assay
ATP hydrolysis was measured using an ATP-regenerating
system as a decrease in A340of NADH at 25 °C The assay
mixture contained 50 mM Mops/KOH buffer (pH 7.0),
50 mM KCl, 2.5 mM phosphoenolpyruvate, 4 mM ATP,
6 mMMgCl2, 200 lgámL)1pyruvate kinase, 200 lgámL)1
lactate dehydrogenase, and 0.2 mMNADH The
spectro-photometer (V-550; Jasco) was equipped with a stirring
device for rapid mixing After a 5-min incubation at 25 °C, a
baseline was monitored for 1 min The reaction was started
by the addition of a3b3c to 1.2 mL of the assay mixture One
unit (U) of activity is de®ned as the activity of 1 lmol ATP
hydrolysedámin)1 The ATPase activities at the initial phase
(the slope of A340between 3 and 13 s) were measured
Fluorescence measurements
Fluorescence was measured with a spectro¯uorometer
(F4500; Hitachi) at 25 °C After nucleotide depletion,
a3b3c was diluted to 100 nMin a cuvette to a total volume
of 1.2 mL in 50 mMMops/KOH (pH 7.0) buffer containing
50 mM KCl and 2 mM MgCl2 The excitation wavelength
was 295 nm and the emission wavelength was 345 nm The slit for excitation was set at 5 nm and the slit for emission at
10 nm The time-course measurements were performed by injecting concentrated MgATP or MgADP solution into the assay mixture to the indicated concentrations while stirring For titration curves, the ¯uorescence intensities were recorded for 3 min after the addition of MgATP solutions Calculation of total Mg2+concentrations
At a high Piconcentration, it is possible that free Mg2+and MgADP concentrations decrease because of complex formation between Pi and Mg2+ It has previously been shown that the activating effects of EDTA and Pion the MgADP-inhibited enzyme are additive, indicating that activation by Pioccurs via a mechanism other than simply reducing the Mg2+concentration [28] However, in order to exclude the possibility of reducing MgADP at high concentrations of Pi, we adjusted the total Mg2+ concen-tration for each Pi concentration in the relevant experi-ments The concentration of each ionic species was calculated as described by Clark et al [29]
R E S U L T S ATPase activity of the a(W463F)3b(Y341W)3c subcomplex after incubation with MgADP
The a(W463F)3b(Y341W)3c subcomplex was preincubated with MgADP at various molar ratios for 10 min The residual ATPase activities at the initial phase were measured by injecting the mixture into the ATPase assay system As shown in Fig 1A,B (®lled circles), the extent of inhibition was almost 100% when the ratio of MgADP to the
a(W463F)3b(Y341W)3c subcomplex reached 1.5 : 1 It has previously been reported that the DNC a3b3c subcomplex was inhibited by MgADP at a 1 : 1 molar ratio [16] In a similar experiment, wild-type a3b3c complex was inhibited
by MgADP at a ratio of 1 : 1.5 (E Muneyuki, unpublished results) Here we conclude that the a(W463F)3b(Y341W)3c subcomplex assumes the MgADP-inhibited form in the same manner as the wild-type a3b3c complex
Effect of Pion the MgADP-inhibited form
We have previously reported that the DNC a3b3c subcom-plex did not exhibit steady-state ATP hydrolysis in the absence of Pi[16], but signi®cant steady-state ATP hydro-lysis was later observed in the presence of Pi[18] Here we examined the effects of Pion the formation of the MgADP-inhibited form of the a(W463F)3b(Y341W)3c subcomplex The extent of ADP inhibition of the a(W463F)3b(Y341W)3c sub-complex after 5 min preincubation with stoichiometric concentrations of MgADP plus various concentrations of potassium phosphate was measured Increasing concentra-tions of Piprevented the formation of the MgADP-inhibited form of the a(W463F)3b(Y341W)3c subcomplex (Fig 2A) The
Pi concentration that yielded half-maximal prevention of inhibition was 21 6.4 mM(mean SEM; Fig 2B), and the extrapolated maximum rate was 69 6.0% of the uninhibited rate, i.e the rate in the absence of ADP KCl (100 mM) in the preincubation medium had no protective effect against MgADP inhibition When the Pi
Trang 3tion was kept at 20 mM and the ratio of MgADP to the
subunit complex in the preincubation mixture was changed,
the extent of inhibition by MgADP also decreased
(Fig 1B) From these results, we conclude that Piprevented
MgADP inhibition under these conditions This is
consis-tent with the previous report that inactivation of F1-ATPase
as a result of the MgADP inhibition could be partially
prevented by Pi [18] The above results indicate that the
a(W463F)3b(Y341W)3c subcomplex has the same properties
for MgADP inhibition and Pi activation as the wild-type
complex, allowing us to investigate the effect of Pi on
MgADP inhibition
Effect of Pion MgADP binding to catalytic sites
Formation of the MgADP-inhibited form is caused by
entrapment of inhibitory MgADP in a catalytic site [5±9]
After prior loading of a catalytic site of MF1[14,30], TF1 [31], and chloroplast F1-ATPase [32] with MgADP, the enzymes hydrolyse ATP with an extended lag phase The protective effect of Pi against ADP inhibition described above may be explained by interference of MgADP binding
by Pi The decrease in the extent of MgADP inhibition, however, does not necessarily mean that ADP does not bind
to the enzyme To clarify this point, MgADP binding to the catalytic sites in the presence of Pi was monitored
by ¯uorescence quenching of the introduced tryptophan
Fig 1 Inhibition of ATPase activity of the a (W463F)3 b (Y341W)3 c
sub-complex by prior incubation with MgADP (A) Time course of ATP
hydrolysis The a (W463F)3 b (Y341W)3 c complex (0.4 l M ) was
preincu-bated at 25 °C for 10 min in the presence of various concentrations of
MgADP Then, 40 lL of the solutions was withdrawn and injected
into 1.2 mL of an ATP assay mixture containing 4 m M ATP The
molar ratio of MgADP to the subcomplex during the preincubation is
shown beside the traces (B) Residual ATPase activity (d) Without P i ;
(j) in the presence of 20 m M potassium phosphate To keep MgADP
concentration constant, MgCl 2 concentrations in the preincubation
mixture were adjusted to be 2 m M and 6 m M in the absence and
presence of P i , respectively The residual ATPase activities at the initial
phase (the slope of A 340 between 3 and 13 s) were plotted against the
molar ratio of MgADP to the subcomplex The speci®c ATPase
activity of enzyme without MgADP, which was set at 100%, was
12.3 Uámg )1 in the absence of potassium phosphate (d) and
29.3 Uámg )1 in the presence of 20 m M potassium phosphate (j).
Fig 2 Prevention of the formation of the MgADP-inhibited form by phosphate (A) Time course of ATP hydrolysis The a (W463F)3 b (Y341W)3 c subcomplex (0.4 l M ) was preincubated at 25 °C for 5 min in the presence of various concentrations of potassium phosphate The P i
concentrations are indicated on the right Then, MgADP (1.2 l M ®nal concentratioin) was added to the preincubation mixture and incubated for a further 5 min Total Mg 2+ concentrations added as MgCl 2 in the preincubation mixture were adjusted to 3.0, 3.5, 4.5, 6.5, 10, and
16 m M for 0, 2.5, 10, 25, 50 and 100 m M potassium phosphate to keep the MgADP concentration constant After the preincubation, 40 lL of the solutions was injected into 1.2 mL of the ATPase assay mixture containing 4 m M ATP The lowest line is a control experiment without ADP or potassium phosphate (B) Dependence of the relative ATP hydrolysis activity of the a (W463F)3 b (Y341W)3 c subcomplex on preloaded potassium phosphate concentration The residual ATPase activities during the initial phase (the slope of A 340 between 3 and 13 s) were measured after preincubation with 1.2 l M MgADP and potassium phosphate as in (A) Relative ATPase activities were calculated by normalizing ATPase activities without preloaded MgADP at each potassium phosphate concentration to 100% The solid line was drawn
by ®tting the equation (a + b[P i ])/(K d + [P i ]) to the data points Here, a is the residual ATPase activity at 1.2 l M ADP in the absence of
P i , and b is the maximum activity regained in the presence of P i K d is the apparent dissociation constant for P i , which was deduced to be
21 m M
Trang 4residues On addition of saturating amounts of nucleotide
(1 mMMgADP or 1 mMMgATP), the ¯uorescence of the
three introduced tryptophans was completely quenched
Figure 3A shows the time course of the ¯uorescence
change on addition of MgADP In the experiments, the
concentrations of the a(W463F)3b(Y341W)3c subcomplex and MgADP were 100 nM and 300 nM, respectively Contrary
to our expectation, the presence of Pihad little effect on the binding of ADP to the catalytic sites Quenching was essentially complete within 20 s Actually, the rate of the MgADP binding was slightly slower at higher Pi concen-trations; however, the magnitude of ¯uorescence quenching was affected little (Fig 3C) in the Piconcentration range in which the protective effect of Piwas most prominent These results suggest that Piprevents MgADP inhibition, although MgADP remains bound to the catalytic sites
Effect of Pion MgATP binding to catalytic sites Previously, we suggested that Piacts at a catalytic site to prevent ADP inhibition [18] However, given that, Pidid not interfere with ADP binding at any of the catalytic sites, there are two possible explanations of our results One is that Pi binds to a catalytic site, but the position is at the c phosphate
of ATP, and does not interfere with ADP binding The other
is that Pibinds to a site other than any of the catalytic sites
In the ®rst case, it is possible that Pibinding interferes in ATP binding, because of the c phosphate To con®rm this possibility, MgATP binding to the catalytic sites was monitored by ¯uorescence quenching of the introduced tryptophan residues in the presence of Pi The time course of the ¯uorescence quenching on addition of MgATP in the presence of various concentrations of Piis shown in Fig 3B Although the ®nal magnitude of the ¯uorescence quenching
by the addition of MgATP did not signi®cantly depend on the Piconcentration (Fig 3C), ATP binding to the catalytic sites was generally slower at higher Pi concentrations However, the result was somewhat ambiguous, possibly because of the hydrolysis of ATP to ADP during the experiment To clarify this point, the a(W463F)3b(E190Q/ Y341W)3c subcomplex, which was able to bind MgATP but unable to hydrolyse MgATP [27], was used In the case of the E coli F1-ATPase, a mutant equivalent to a3b(E190Q/ Y341W)3c displayed a nucleotide-binding pattern that was similar to that of the a3b(Y341W)3c subcomplex [24] Figure 4B demonstrates the time course of ATP binding
to the a(W463F)3b(E190Q/Y341W)3c complex in the presence of
Pi Compared with Fig 3B, Fig 4B indicates more clearly that ATP bound to the catalytic sites more slowly at higher
Piconcentrations Two time constants were estimated by
®tting a double-exponential function to the data and plotted against Piconcentration (Fig 4C) The time resolution of our experimental system was, however, not good enough for the fast phase and we did not attempt to analyze it On the other hand, the slow time constant of MgATP binding was clearly longer at higher Pi concentrations The apparent af®nity of Piwas estimated to be 19 3.6 mMby ®tting a simple Michaelis±Menten equation to the data for the slow time constant This value was similar to the Kdof 21 mM measured for suppression of MgADP inhibition (Fig 2B)
On the other hand, the presence of Pihad little in¯uence on the binding of ADP to the catalytic sites (Fig 4A,D) Again,
Pihad little effect on the time constant or magnitude of
¯uorescence quenching by MgADP binding in the Pi concentration range where the effect of Pi was most prominent for the a(W463F)3b(Y341W)3c complex From these results, we conclude that Piis bound at a catalytic site of the
a(W463F)3b(Y341W)3c subcomplex where it competes with
Fig 3 Eect of P i on ADP binding to the a (W463F)3 b (Y341W)3 c
sub-complex (A) Time course of MgADP binding to the a (W463F)3
b (Y341W)3 c subcomplex The reaction mixture contained 50 m M Mops/
KOH (pH 7.0), 50 m M KCl, 100 n M a (W463F)3 b (Y341W)3 c, and the
indicated concentrations of potassium phosphate and MgCl 2 MgCl 2
concentrations were adjusted as in Fig 2 MgADP (300 n M ) was
added at time zero (B) Time courses of the ¯uorescence quenching on
addition of MgATP The experimental procedure is the same as in (A),
except that MgATP was added instead of MgADP (C) The amplitude
of ¯uorescence quenching upon addition of MgATP (d) and MgADP
(j) plotted against P i concentration The amplitudes were read from
(A) and (B).
Trang 5MgATP Close coincidence of the apparent af®nity of Pi
that interferes with ATP binding (19 mM) and the apparent
af®nity that prevents formation of the MgADP-inhibited
form (21 mM) suggests that the Pithat prevents MgADP
inhibition is located in a catalytic site even though it does not
signi®cantly affect ADP binding to that catalytic site
Titration of MgATP binding
to the a(W463F)3b(E190Q/Y341W)3c complex
Figure 5A shows MgATP binding to the a(W463F)3b(E190Q/
Y341W)3c complex in the presence or absence of 10 mM
phosphate As shown in Fig 5A,B, MgATP binding was
fast in the absence of phosphate, but slow in the presence of
10 mM phosphate at appropriate MgATP concentrations
At 200 nM MgATP, the binding was slowest When
MgATP was lower or higher than 200 nM, the MgATP
binding was fast (Fig 5A,C) At 1 lM, the rate of MgATP
binding in the presence of Piwas faster than at 300 nM This
increase in the rate of ATP binding may simply be due to the
high concentration of ATP When MgATP concentration
was lower than 100 nM, the rate of MgATP binding was
dif®cult to estimate because the ¯uorescence intensity
change was small Nevertheless, it seems that MgATP
binding is most affected by Piunder bi-site conditions where
MgATP binds to the second catalytic site of the enzyme
The above results suggest that Piinhibits ATPase activity,
particularly at low ATP concentrations We then compared
the initial ATPase activity of the a(W463F)3b(Y341W)3c
com-plex in the presence and absence of 20 mM Pi Although
there was not a precise agreement with the data in Fig 5C,
which was obtained using the a(W463F)3b(E190Q/Y341W)3c
complex, the ATPase activity was indeed 29, 48, and 13% inhibited at 0.3, 1, and 10 lMATP in the presence of Pi At
100 lMATP, there was no apparent inhibition by Pi
D I S C U S S I O N
Pi suppresses formation of the MgADP-inhibited form There may be several explanations The simplest is that Pi binds to a catalytic site, and inhibitory ADP is ejected from
it The second is that Piand ADP bind simultaneously to the catalytic site and the enzyme is kept active The third possibility is that Pibinds to noncatalytic sites (or any other site) while ADP binds to the catalytic site The fourth possibility is that Pibinds to the noncatalytic sites (or any other site) and inhibitory ADP is ejected from the catalytic sites The last possibility is reminiscent of the situation in which ATP binding to the noncatalytic sites activates F1 from the MgADP-inhibited form [12±15] Two important points are whether ADP remains bound to the catalytic sites when Pi is added to activate the ADP-inhibited enzyme, and the location of Pibinding When Piwas added
to the assay mixture, however, the effect of Piin relieving MgADP inhibition was not clear This may be because Piin the assay mixture simultaneously acts as competitive inhibitor of ATP hydrolysis and suppresser of MgADP inhibition In the case of EF1, Piwas shown to inhibit uni-site and bi-uni-site ATP hydrolysis [33] Therefore, we mainly examined the effect of Pion MgADP inhibition by adding
Pito the preincubation mixture with ADP and observing the resultant ATPase activity and ¯uorescence quenching From our results, we conclude that the presence of Piat a catalytic site interferes with the formation of the
MgADP-Fig 4 Time-course of MgATP/MgADP binding to the a (W463F)3 b (E190Q/Y341W)3 c subcomplex (A, B) Time course of MgADP (A)/MgATP (B) binding to the a (W463F)3 b (E190Q/Y341W)3 c subcomplex in the presence of P i The reaction mixture contained 50 m M Mops/KOH (pH 7.0), 50 m M
KCl, 100 n M a (W463F)3 b (E190Q/Y341W)3 c, and the indicated concentrations of potassium phosphate and MgCl 2 MgCl 2 concentrations were adjusted
as in Fig 2 MgADP or MgATP (300 n M ) was added at 0 s (C) Dependence of the time constant of the MgATP binding (s) to the a (W463F)3 b (E190Q/ Y341W)3 c subcomplex on P i concentration The time constants were estimated by ®tting a double-exponential function to the time course (j) Fast time constants; (h) slow time constants The solid line was drawn by ®tting the equation a[P i ]/(K d + [P i ]) to the data points Here, a is the maximum time constant in the presence of P i , and K d is the apparent dissociation constant for P i , which was deduced to be 19 m M (D) The amplitude of ¯uorescence quenching on addition of MgATP (d) and MgADP (j) plotted against P i concentration.
Trang 6inhibited form even while MgADP is still bound at that
catalytic site
It is known that a number of anions exert various
complex effects on F1-ATPase [34±37] Potentially, some of
these effects are the result of anion binding to sites other
than the catalytic nucleotide-binding sites However, in the
present case in which Piprevents MgADP inhibition, it is
highly likely that the presence of Pi at a catalytic site
suppresses the formation of the MgADP-inhibited state
The recently reported crystal structure of mitochondrial F1
-ATPase with nucleotide bound to all three catalytic sites
shows that sulfate, which is an analog of phosphate, indeed
binds near the c phosphate position of ATP at a catalytic site [38] As previously proposed [18], the presence of Piat a catalytic site may shift the equilibrium from the MgADP-inhibited form to the enzyme±MgADP±Picomplex, which is
an active intermediate in the catalytic cycle Maximum protection from ADP inhibition by Pi was only 69% (Fig 2B), suggesting that there may be other inactive enzyme±MgADP±Picomplexes, and, even in the presence of excess Pi, the equilibrium cannot be shifted completely to the active complex In the case of membrane-bound ATP synthase, membrane energization can convert the inactive form of the enzyme into the active form [39] A combination
of Pi and membrane energization may fully protect the enzyme from ADP inhibition under phosphorylating con-ditions The apparent Kd of Pi that interferes with ATP binding (19 mM), or that which prevents formation of the MgADP-inhibited form (21 mM), was somewhat higher than the apparent Kmfor ATP synthesis by thermophilic
FoF1-ATPase reconstituted into proteoliposomes (6±9 mM) [17] This discrepancy may re¯ect the difference in the experimental conditions, or the membrane potential may cause some increase in the af®nity for Pi
In previous studies, using the ¯uorescence quenching of genetically introduced tryptophans, competition of MgATP binding to the catalytic sites [40] or noncatalytic sites [41] with Pi was not observed Our result differs from the previous data, but this apparent discrepancy is probably due
to differences in the experimental conditions For example, using 0.1 lM a(W463F)3b(Y341W)3c and 0.3 lMMgATP, the
®nal ¯uorescence level induced by MgATP binding was not signi®cantly in¯uenced by the presence or absence of Pi (Fig 3B,C) Only a Pi-dependent change in the rate of MgATP binding was detected (Fig 3B) Unfortunately, the result was somewhat unclear, probably because of hydro-lysis of MgATP during the experiment, therefore we used the a(W463F)3b(E190Q/Y341W)3c complex, which is unable to hydrolyse ATP The ®nal ¯uorescence level quenched by MgATP binding in this case was also almost independent of
Pi (Fig 4B,D) The retardation of ATP binding in the presence of Piwas clearly exhibited (Figs 4B,C) and it was found that this retardation was observed over a limited ATP concentration range (0.1 lM< [ATP] < 1 lM, Fig 5A,C) The inhibition of ATPase activity at low ATP concentration by 20 mMPiis qualitatively consistent with the retarded ATP binding Below 0.1 lM ATP, the low signal to noise ratio in the ¯uorescence measurement does not allow a ®rm conclusion, but it seems that ATP binding
to the ®rst catalytic site (uni-site) is not signi®cantly affected
by the presence of Pi In the concentration range in which retardation of ATP binding by Pi was observed, ATP binding to the second catalytic site (bi-site) occurred It is tempting to conclude that Pi competes with ATP at the second catalytic site It may imply that the ®rst site favors ATP binding whereas the second site favors Pi binding Preferred binding of substrate or product at catalytic sites in different conformations has been suggested by Boyer [42]
At higher ATP concentrations, the effect of Pion the ATP-binding rate was not observed At high ATP concentrations, however, ATP binding is inherently fast, and the apparent absence of the effect of Pion the ATP binding rate may be due to the limited time resolution of our experimental system Measurements with high time resolution will provide more detailed information on nucleotide binding
Fig 5 Fluorescence titration of the catalytic sites of the
a (W463F)3 b (E190Q/Y341W)3 c mutant with MgATP with and without
phosphate (A) Time course of MgATP binding in the presence of
10 m M phosphate The indicated concentrations of MgATP were
added at 0 s (B) Time course of MgATP binding in the absence of P i
MgATP was added as in (A) at 0 s (C) Dependence of the time
constant of MgATP binding to the a (W463F)3 b (E190Q/Y341W)3 c
sub-complex on MgATP concentrations in the presence of P i The time
constants were estimated by ®tting a double-exponential function to
the time course of MgATP binding (d) Fast time constants; (j) slow
time constants.
Trang 7A C K N O W L E D G E M E N T S
We thank Drs T Hisabori, H Noji, D Bald, Y Kato-Yamada,
T Masaike and Y Hirono-Hara for helpful discussion We also thank
Dr J Hardy for critical reading of the manuscript.
R E F E R E N C E S
1 Boyer, P.D (1997) The ATP synthase: a splendid molecular
machine Annu Rev Biochem 66, 717±749.
2 Boyer, P.D (1993) The binding change mechanism for ATP
synthase some probabilities and possibilities Biochim Biophys.
Acta 1140, 215±250.
3 Muneyuki, E., Noji, H., Amano, T., Masaike, T & Yoshida, M.
(2000) F o F 1 -ATP synthase: general structural features of
ÔATP-engineÕ and a problem on free energy transduction Biochim.
Biophys Acta 1458, 467±481.
4 Abrahams, J.P., Leslie, A., Lutter, R & Walker, J.E (1994)
Structure at 2.8 AÊ resolution of F 1 -ATPase from bovine heart
mitochondria Nature (London) 370, 621±628.
5 Moyle, J & Mitchell, P (1975) Active/inactive state transitions of
mitochondrial ATPase molecules in¯uenced by Mg 2+ , anions and
aurovertin FEBS Lett 56, 55±61.
6 Fitin, A.F., Vasilyeva, E.A & Vinogradov, A.D (1979) An
inhibitory high anity binding sites for ADP in the
oligomycin-sensitive ATPase of beef heart submitochondrial particles
Bio-chem Biophys Res Commun 86, 434±439.
7 Vasilyeva, E.A., Minkov, I.B., Fitin, A.F & Vinogradov, A.D.
(1982) Kinetic mechanism of mitochondrial adenosine
triphos-phatase Biochem J 202, 1±14.
8 Vasilyeva, E.A., Minkov, I.B., Fitin, A.F & Vinogradov, A.D.
(1982) Kinetic mechanism of mitochondrial adenosine
triphos-phatase Biochem J 202, 15±23.
9 Drobinskaya, I.Y., Kozlov, I.A., Murataliev, M.B & Vulfson,
E.N (1985) Tightly bound adenosine diphosphate, which inhibits
the activity of mitochondrial F 1 -ATPase, is located at the catalytic
site of the enzyme FEBS Lett 182, 419±424.
10 Milgrom, Y.M & Boyer, P.D (1990) The ADP that binds tightly
to nucleotide-depleted mitochondrial F 1 -ATPase and inhibits
catalysis is bound at a catalytic site Biochim Biophys Acta 1020,
43±48.
11 Muneyuki, E., Makino, M., Kamata, H., Kagawa, Y., Yoshida,
M & Hirata, H (1993) Inhibitory eect of NaN 3 on the F o F 1
ATPase of submitochondrial particles as related to nucleotide
binding Biochim Biophys Acta 1144, 62±68.
12 Milgrom, Y.M., Ehler, L.L & Boyer, P.D (1990) ATP binding at
noncatalytic sites of soluble chloroplast F 1 -ATPase is required for
expression of the enzyme activity J Biol Chem 265, 18725±
18728.
13 Milgrom, Y.M., Ehler, L.L & Boyer, P.D (1991) The
charac-teristic and eect on catalysis of nucleotide binding to noncatalytic
sites of chloroplast F 1 -ATPase J Biol Chem 266, 11551±11558.
14 Jault, J.M & Allison, W.S (1993) Slow binding of ATP to
noncatalytic nucleotide binding sites which accelerates catalysis is
responsible for apparent negative cooperativity exhibited by the
bovine mitochondrial F 1 -ATPase J Biol Chem 268, 1558±1566.
15 Hyndman, D.J., Milgrom, Y.M., Bramhall, E.A & Cross, R.L.
(1994) Nucleotide binding sites on Escherichia coli F 1 -ATPase
speci®city of noncatalytic sites and inhibition at catalytic sites by
MgADP J Biol Chem 269, 28871±28877.
16 Matsui, T., Muneyuki, E., Honda, M., Allison, W.S., Dou, C &
Yoshida, M (1997) Catalytic activity of the a 3 b 3 c complex of
F 1 -ATPase without noncatalytic nucleotide binding sites J Biol.
Chem 272, 8215±8221.
17 Bald, D., Amano T., Muneyuki, E., Pitard, B., Rigaud, J.L.,
Kruip, J., Hisabori, T., Yoshida, M & Shibata, M (1998) ATP
synthesis by F o F 1 -ATP synthase independent of noncatalytic
nucleotide binding sites and insensitive to azide inhibition J Biol Chem 273, 865±870.
18 Bald, D., Muneyuki, E., Amano T., Kruip, J., Hisabori, T & Yoshida, M (1999) The noncatalytic site-de®cient a 3 b 3 c sub-complex and F o F 1 -ATP synthase can continuously catalyse ATP hydrolysis when P i is present Eur J Biochem 262, 563±568.
19 Dou, C., Grodsky, N.B., Matsui, T., Yoshida, M & Allison, W.S (1997) ADP-¯uoroaluminate complexes are formed cooperatively
at two catalytic sites of wild-type and mutant a 3 b 3 c subcomplexes
of the F 1 -ATPase from the thermophilic Bacillus PS3 Biochem-istry 36, 3719±3727.
20 Minkov, I.B & Strothmann, H (1989) The eect of azide on regulation of the chloroplast H + -ATPase by ADP and phosphate Biochim Biophys Acta 973, 7±12.
21 Yalamova, M.V., Vasilyeva, E.A & Vinogradov, A.D (1982) Mutually dependent in¯uence of ADP and P i on the activity
of mitochondrial adenosine triphosphatase Biochem Int 4, 337± 344.
22 Weber, J., Wilke-Mounts, S., Lee, R.S.F., Grell, E & Senior, A.E (1993) Speci®c placement of tryptophan in the catalytic sites of Escherichia coli F 1 -ATPase provides a direct probe of nucleotide binding: maximal ATP hydrolyses occurs with three sites occu-pied J Biol Chem 268, 20126±20133.
23 Dou, C., Fortes, P.A.G & Allison, W.S (1998) The
a 3 (bY341W) 3 c subcomplex of the F 1 -ATPase from the thermo-philic Bacillus PS3 fails to dissociate ADP when MgATP is hy-drolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP Biochemistry
37, 16757±16764.
24 Weber, J., Hammond, S.T., Wilke-Mounts, S & Senior, A.E (1998) Mg 2+ coordination in catalytic sites of F 1 -ATPase Bio-chemistry 37, 608±614.
25 Yanisch-Perron, C., Vieira, J & Messing, J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors Gene 33, 103±119.
26 Matsui, T & Yoshida, M (1995) Expression of the wild-type and the Cys-/Trp-less a 3 b 3 c complex of thermophilic F 1 -ATPase in Escherichia coli Biochim Biophys Acta 1231, 139±146.
27 Amano, T., Tozawa, K., Yoshida, M & Murakami, H (1994) Spatial precision of a catalytic carboxylate of F 1 -ATPase b subunit probed by introducing dierent carboxylate-containing side chains FEBS Lett 348, 93±98.
28 Bulygin, V.V & Vinogradov, A.D (1991) Interaction of Mg 2+
with F 0 F 1 mitochondrial ATPase as related to its slow active/ inactive transition Biochem J 276, 149±156.
29 Clark, D.D., Daggett, S.G & Schuster, S.M (1984) Pre-steady-state kinetics of beef heart mitochondrial ATPase Arch Biochem Biophys 233, 378±392.
30 Chernyak, B.V & Cross, R.L (1992) Adenine nucleotide binding sites on mitochondrial F 1 -ATPase: studies of the inactive complex formed upon binding ADP at catalytic site Arch Biochem Biophys 295, 247±252.
31 Yoshida, M & Allison, W.S (1986) Characterization of the catalytic and noncatalytic ADP binding sites of the F 1 -ATPase from the thermophilic bacterium, PS3 J Biol Chem 261, 5714± 5721.
32 Feldman, R.I & Boyer, P.D (1985) The role of tightly bound ADP on chloroplast ATPase J Biol Chem 260, 13088±13094.
33 Muneyuki, E., Yoshida, M., Bullough, D.A & Allison, W.S (1991) Heterogeneous hydrolysis of substoichiometric ATP by the
F 1 -ATPase from Escherichia coli Biochim Biophys Acta 1058, 304±311.
34 Ebel, R.E & Lardy, H.A (1975) Stimulation of rat liver mito-chondrial adenosine triphosphatase by anions J Biol Chem 250, 191±196.
35 Recktenwald, D & Hess, B (1977) Allosteric in¯uence of anions
on mitochondrial ATPase of yeast FEBS Lett 76, 25±28.
Trang 836 Jault, J.-M., Di Pietro, A., Falson, P., Gautheron, D.C &
Gof-feau, A (1989) A yeast strain with mutated b-subunits of
mito-chondrial ATPase-ATPsynthase: high azide and bicarbonate
sensitivity of the ATPase activity Biochem Biophys Res
Com-mun 158, 392±399.
37 Wong, S.-Y., Matsuno-Yagi, A & Hate®, Y (1984) Kinetics of
ATP hydrolysis by F 1 -ATPase and the eects of anion activation,
removal of tightly bound nucleotides, and partial inhibition
of the ATPase by covalent modi®cation Biochemistry 23, 5004±
5009.
38 Menz, R.I., Walker, J.E & Leslie, A.G.W (2001) Structure of
bovine mitochondrial F 1 -ATPase with nucleotide bound to all
three catalytic sites: implications for the mechanism of rotary catalysis Cell 106, 331±341.
39 Galkin, M.A & Vinogradov, A.D (1999) Energy-dependent transformation of the catalytic activities of the mitochondrial Fo x
F 1 -ATP synthase FEBS Lett 448, 123±126.
40 LoÈau, S., Weber, J & Senior, A.E (1998) Catalytic site nucleotide binding and hydrolysis in F 1 F o -ATP synthase Biochemistry 37, 10846±10853.
41 Weber, J & Senior, A.E (1995) Location and properties of pyrophosphate binding sites in Escherichia coli F 1 -ATPase J Biol Chem 270, 12653±12658.
42 Boyer, P.D (2001) Biokhimiya, in press.