From the
The dissociation constant
( K
The F
The
dissociation constant of 10
However, a dissociation constant of 10
Materials
A summary of the specific activities of the submitochondrial
particles used in these experiments is shown in Table I. The ATPase
activity of ETPH(Mg
The dissociation constants for ATP,
K
This study describes changes in the affinity of the
membrane-bound F
The rate of the
energy-dependent dissociation of ATP bound in high affinity catalytic
sites of F
Among the most
interesting observations reported in this paper are the changes in
standard free energy accompanying ATP dissociation from high affinity
catalytic sites of membrane-bound F
These values for
K
The rate of
ATP hydrolysis by KCl-washed ETPH(Mg
It is noteworthy that the kinetics of ATP binding and release
by the two types of particles under energized and non-energized
conditions were essentially the same (). It should be
emphasized, however, that under the experimental conditions described
in this paper 1 mg of KCl-washed ETPH(Mg
It is clear from the
experiments of that energization brought about a
reduction of almost 10-fold in the rate of binding of ATP in high
affinity catalytic sites. This would seem intuitively reasonable since
under energized conditions the system is poised to dissociate ATP
rather than bind it. It should be pointed out, however, that the on
rates shown in for ETPH(Mg
The question
may arise whether heterogeneity in catalytic sites might influence the
kinetic constants determined in this paper. shows that the
ATPase activity of the submitochondrial particles used in these
experiments was inhibited at least 90% by oligomycin. If 10% of the
membrane-bound enzyme was non-energizable but participated in ATP
binding, that binding would be rapid (10
A variety of observations suggest that the ATP on
rates reported in this study are a reasonable measure of the rate of
ATP binding in catalytic sites of membrane-bound F
In summary, this paper
shows that the energy-dependent dissociation of ATP bound in high
affinity catalytic sites of submitochondrial particles received
contributions from two important variables. First, proton flux which
brought about a 10
ETPH(Mg
Preparation of submitochondrial particles and the conditions used
for each of the measurements are described under ``Experimental
Procedures.''
ATP synthesis
was measured at the temperatures shown using the protocol described
under ``Experimental Procedures.''
We thank R. L. Cross and J. D. Robinson for useful
discussions during the preparation of the manuscript. Marcus Hutcheon
provided excellent technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) for ATP bound in the
high affinity catalytic site of membrane-bound beef heart mitochondrial
ATPase (F
) was calculated from the ratio of the rate
constants for the reverse dissociation step
( k
) and the forward binding step
( k
). k
for ATP
bound to submitochondrial particles or to submitochondrial particles
washed with KCl so as to activate ATPase activity was accelerated by
about five orders of magnitude during respiratory chain-linked
oxidations of NADH. In the presence of NADH and 0.1 m
M ADP,
k
increased more than six orders of
magnitude. These energy-dependent dissociations of ATP were sensitive
to the uncoupler carbonyl cyanide
p-trifluoromethyloxyphenylhydrazone. Only small changes in
k
were observed in the presence of NADH or
NADH and ADP. K
at 23
°C in the absence of NADH and ADP was 10
M, in the presence of NADH, 3 µ
M, and in
the presence of NADH and 0.1 m
M ADP, 60 µ
M. Thus,
the dissociation of ATP during the transition from non-energized to
energized states was, under these conditions, accompanied by observed
free energy changes of 8 and 9.7 kcal/mol, respectively.
F
(
)
-ATPase
catalyzes the terminal transphosphorylation reaction of oxidative
phosphorylation, that is, the formation of ATP from ADP and
P
. The required energy for the reaction, stored as a proton
motive force (Mitchell, 1961), is supplied by oxidations in the
respiratory chain. Although the coupling reaction, that is, the manner
in which proton flux is utilized to drive ATP formation, is only poorly
understood, progress has been made in elucidating the mechanism of
action of the mitochondrial ATPase in catalyzing ATP synthesis (for
reviews see Penefsky and Cross, 1991; Hatefi, 1993). The elementary
steps in the hydrolysis of ATP by both the soluble (Grubmeyer et
al., 1982; Cross et al., 1982) and membrane-bound forms
of beef heart mitochondrial F
(Penefsky, 1985a, 1985b)
supported a model for ATP synthesis in oxidative phosphorylation in
which ATP could be formed from bound ADP and P
with little
or no change in free energy (K
for the catalytic step was
near unity). The free energy of binding of product ATP in high affinity
catalytic sites of F
was viewed as the driving force for
ATP formation (K
for the binding step was 10
M
). Thus, the major requirement for
energy in oxidative phosphorylation was for the dissociation of product
ATP from high affinity catalytic sites (Penefsky and Cross, 1991).
Insight into the nature of the coupling device was provided by further
observations suggesting long range interactions, apparently involving
the transmission of conformational changes, between proton-binding
sites in F
and the catalytic sites of F
(Penefsky, 1985c; Matsuno-Yagi et al., 1985). These
observations support and extend the original proposals of Boyer (1993)
in the binding change mechanism of oxidative phosphorylation.
M for
dissociation of ATP from high affinity catalytic sites of soluble
(Grubmeyer et al., 1982) or non-energized membrane-bound
F
(Penefsky, 1985a) suggested that during oxidative
phosphorylation the catalytic sites would have to undergo a decrease in
affinity for ATP of many orders of magnitude. Experimental precedent
for cyclic changes in the affinity of catalytic sites was established
in studies of the hydrolysis of trinitrophenyl-ATP (TNP-ATP) by soluble
F
. TNP-ATP and TNP-ADP both were bound at site 1 of the
enzyme with an affinity too high to be measured. Nevertheless, the
enzyme catalyzed the hydrolysis of TNP-ATP (Grubmeyer and Penefsky,
1981a, 1981b).
M is equivalent to a standard free energy change of
about 16 kcal/mol, whereas the standard free energy change accompanying
ATP hydrolysis was reported to be about -8.5 kcal/mol (Rosing and
Slater, 1972) and the phosphorylation potential generated by respiring
submitochondrial particles (
G
) was reported
to be 10.6 kcal/mol (Ferguson and Sorgato, 1977). This apparent
discrepancy is resolved by observations presented in this paper that
the dissociation constant for ATP
( K
) from submitochondrial
particles under conditions of oxidative phosphorylation used in these
experiments was about 60 µ
M. Thus, the observed free
energy change relevant to the dissociation of ATP during the transition
from non-energized to energized states was 9.7 kcal/mol. The changes in
affinity for ATP were expressed largely as an increase in the rate of
ATP dissociation under energized conditions. It was found that the rate
of dissociation of ATP was accelerated five orders of magnitude in the
presence of NADH and about six orders of magnitude in the presence of
NADH and 0.1 m
M ADP. The observed energized dissociation
constant of 3 µ
M (in the presence of NADH alone) is in the
same range as values reported for the chloroplast thylakoid of 7
µ
M (Magnusson and McCarty, 1976) and for Paracoccus
denitrificans of 16 µ
M (Perez and Ferguson, 1990a,
b).
P
(enzyme grade) was purchased from ICN and
used without further purification. [
-
P]ATP
was prepared as described (Glynn and Chappel, 1964). The specific
radioactivity of most preparations was 5
10
counts/min/nmol. Hexokinase, ATP, ADP, and bovine serum albumin
were purchased from Sigma. NADH (``100% pure'') was purchased
from Boehringer Mannheim. Methods Submitochondrial particles, ETPH(Mg
), were prepared
from beef heart mitochondria (Beyer, 1967) and activated by washing
with buffered solutions of KCl (Penefsky, 1985a). The specific ATPase
activity of preparations of submitochondrial particles was about
0.8-1.0 units/mg of protein and that of KCl-washed particles 10
units/mg of protein. The amounts of
P
and
[
-
P]ATP in reaction mixtures were
determined by scintillation counting of organic and aqueous phases
after extraction of perchloric acid-quenched samples with
isobutanol/benzene (Lindberg and Ernster, 1956). Aliquots of each of
the phases were added to a scintillation mixture described earlier
(Penefsky, 1977) and counted in a Beckman LS-6500 liquid scintillation
counter. F
(Penefsky, 1977) and OSCP (Senior, 1979) were
prepared as described. The preparation of OSCP was carried out through
the step of extraction with ammonia. The resulting supernatant solution
was treated with 42% solid ammonium sulfate, and the precipitate was
collected and dissolved with 20 ml of a solution containing 50 m
M Tris-SO
, pH 8, 0.1 m
M dithiothreitol, and 0.1
m
M EDTA. The solution was stored at -70 °C. After
thawing, the solution was centrifuged at 165,000
g to
remove the precipitate that formed. The supernatant was stored at
-70 °C and dialyzed before use versus a solution
containing 50 m
M Tris-SO
, pH 8, 0.1 m
M dithiothreitol, and 0.1 m
M EDTA.
Reconstitution of KCl-washed Particles with Soluble
F
F(3.7 mg), OSCP (2.8 mg), and albumin
(35 mg) were incubated with 44 mg of KCl-washed
ETPH(Mg
) in a buffer containing 0.25
M sucrose, 50 m
M Tris acetate, pH 7.4, 12 m
M P
, pH 7.4, 5 m
M ATP, 1 m
M dithiothreitol, 0.5 m
M EDTA, and 20 m
M succinate
for 5 min at room temperature with rapid stirring. The final volume was
17.5 ml. Succinate was essential for maximal and stable reconstitution.
The reconstituted particles were separated from the soluble ATPase by
centrifugation for 20 min at 40,000 revolutions/min in a Beckman Ti
50.2 rotor (4 °C). The pellets were washed once by resuspension in
15 ml of a buffer containing 0.25
M sucrose, 5 m
M MgSO
, 40 mg of albumin, and 1 m
M dithiotheitol, followed by centrifugation under the same
conditions, and resuspended at 15 mg/ml in the same buffer. Energy-dependent Dissociation of [
-
P]ATP
Bound in High Affinity Catalytic Sites of Submitochondrial
Particles-The reaction mixture contained 20 m
M Tris-SO
, pH 8, 5 m
M P
, 5 m
M MgSO
, 0.25
M sucrose, 50 m
M glucose,
0.1 µ
M [
-
P]ATP, 200 units of
hexokinase, 1 m
M NADH, and 1-2 mg of the particles. The
final volume was 1.0 ml. The particles were incubated for 1 min at room
temperature with all additions except NADH, hexokinase, and radioactive
ATP. After incubation for 5-10 s with
[
-
P]ATP (in order to form the
enzyme-substrate complex), hexokinase was added and an additional 5 s
of incubation was allowed to convert any free
[
-
P]ATP to glucose-6-
P. In a
separate experiment, 5 s of incubation in the presence of 200 units of
hexokinase was sufficient to convert all added
[
-
P]ATP to glucose-6-
P.
Respiration was initiated with NADH. The reaction was terminated at the
times indicated in the figures by adding 1.0 ml of 2
N HCl.
[
-
P]ATP and
P
were
determined by scintillation counting of aqueous and organic phases
after extraction of samples with isobutanol/benzene as described by
Penefsky (1977). Glucose-6-
P was determined by the same
methods after hydrolysis of the samples in 1
N HCl at 100
°C for 7 min. The amount of [
-
P]ATP
bound in catalytic sites at each time point was determined by carrying
out a cold chase measurement as described under Measurement of Unisite
Catalysis.
Measurement of Unisite Catalysis
Submitochondrial
particles (the type and amounts indicated in the figure legends) were
mixed with substoichiometric amounts of
[-
P]ATP in a reaction mixture containing 20
m
M Tris-SO
, pH 8, 5 m
M P
, 5
m
M MgSO
, and 0.25
M sucrose. The final
volume was 1.0 ml. The reaction was carried out at room temperature in
a flat bottom, glass scintillation vial with rapid stirring via a
magnetic stirring bar and was started by adding the
[
-
P]ATP. At the times indicated in the
figures, the reaction was stopped by adding 1 ml of 2
N HCl
(acid quench), or a cold chase was carried out by adding 40 µl of
150 m
M MgATP, permitting the reaction to proceed for an
additional 5 s and then quenching with HCl as before. The amounts of
[
-
P]ATP and
P
in
reaction mixtures were determined by scintillation counting of aqueous
and organic phases after extraction of deproteinized reaction mixtures
with isobutanol/benzene. In separate experiments, it was determined
that the extent of hydrolysis of the cold chase in these experiments
was less than 2% of the added nonradioactive ATP. Thus, no correction
was made for cold chase hydrolysis. Corrections were made for the
1-2% of free
P
present in the
[
-
P]ATP.
Measurement of ATP Binding in High Affinity Catalytic
Sites of the Membrane-bound Enzyme
Binding was determined using
acid quench and cold chase techniques as described earlier (Penefsky,
1985a). Reconstituted, KCl-washed ETPH(Mg), 0.25 mg,
or 2 mg of ETPH(Mg
), were mixed with 0.01 nmol or 0.1
nmol, respectively, of [
-
P]ATP, and the
enzyme-substrate complex was allowed to form for periods of time
indicated on the abscissae before the reactions were quenched with acid
(lower curves) or chased by an excess of cold ATP (upper curves). The
difference between hydrolysis in the acid quench and hydrolysis in the
cold chase is set equal to the amount of radioactive ATP bound in
catalytic sites. The amount of
P
formed in
cold chase experiments (upper curves) was set equal to the amount of
enzyme-substrate complex formed (F
P).
This latter value was used to calculate the bimolecular rate constants
for ATP binding. The results are plotted in the form of a second order
rate equation, as explained in the legends to Fig. 1and 2.
Figure 1:
Rate of binding of
[-
P]ATP in catalytic sites of
reconstituted, KCl-washed ETPH(Mg
). A, 0.125
mg of the particles were incubated for 1 min at room temperature in a
buffer containing 20 m
M Tris-SO
, pH 8, 5 m
M KH
PO
, 5 m
M MgSO
, and
0.25
M sucrose. The final volume was 1.0 ml. The reaction was
started by adding 0.005 nmol of [
-
P]ATP.
The acid quench ( lower curves) represents the amount of
P
formed after the indicated seconds of
incubation. The cold chase ( upper curves) represents the
amount of
P
formed plus the amount of
[
-
P]ATP hydrolyzed following addition of 6
m
M MgATP. After addition of the cold chase, the enzyme was
allowed to turn over for 5 s before the reaction was stopped by
addition of 1.0 ml of 2
N HCl. In the acid quench experiment,
1.0 ml of 2
N HCl was added before the nonradioactive ATP.
P
formed was separated from
[
-
P]ATP as described under
``Experimental Procedures.'' Hydrolysis is expressed as the
percent of the total [
-
P]ATP added. Low
concentrations of enzyme and [
-
P]ATP were
used in panel A to permit a better resolution of the initial
portion of the on reaction. For the experiments in panels
B-D, the reaction mixtures contained 0.25 mg of particles
and 0.01 nmol of [
-
P]ATP. B, the
reaction mixture was the same as that described for panel A,
except that 0.25 mg of the particles and 0.01 nmol of
[
-
P]ATP were used, and 0.1 m
M ADP
was present in the incubation mixture. C, the reaction mixture
was the same as that described for B except that ADP was not
added, and respiration was started by adding 1 m
M NADH,
followed 10 s later by addition of 0.01 nmol of
[
-
P]ATP. D, the reaction mixture
was the same as that described for C except that 0.1 m
M ADP also was present in the reaction mixture. E,
graphical determination of k
, the
bimolecular rate constants for [
-
P]ATP
binding shown in panels B-E. The constants were
calculated from the slopes by using the equation: k =
slope/F
-ATP
. F
and
ATP
represent the initial concentrations of F
and ATP, respectively. F
P is the
concentration of F
P complex at time
t. The concentration of F
in reaction mixtures was
calculated on the assumption that each milligram of submitochondrial
particles contained 0.4 nmol of F
(Harris et al.,
1977; Beltran et al., 1986; Matsuno-Yagi and Hatefi, 1988).
The designations of each of the slopes refer to panels
A-D. The calculated rate constant in the absence of ADP and
NADH was 3.5
10
M
s
; in the presence of 0.1 m
M ADP, 8
10
M
s
; in the presence of 1 m
M NADH, 8.6
10
M
s
, and in the presence of both ADP and NADH,
4.1
10
M
s
.
Measurement of Oxidative Phosphorylation
The
reaction mixture contained 50 m
M Tris acetate, pH 7.5, 0.25
M sucrose, 50 m
M glucose, 5 m
M magnesium
acetate, 0.4 m
M EDTA, 20 m
M potassium phosphate,
P
(100-300 counts/min/nmol), 1 m
M ADP, 50 units of hexokinase, and 60-70 µg of
submitochondrial particles. After 5 min of incubation at 30 °C, the
reaction was initiated by adding 1 m
M NADH. The final volume
was 0.65 ml. The reaction was stopped by adding 0.05 ml of 70%
perchloric acid. Glucose-6-
P formed was determined after
extraction with isobutanol/benzene as described (Penefsky, 1977).
Negligibly small amounts of glucose-6-
P were formed during
the 5-min preincubation period. The rate of oxidation of NADH was
measured polarographically using a Yellow Springs Instruments model 53
device under exactly the same conditions used for the ATP synthesis
measurement except that
P
was omitted and the
final volume of the reaction mixture was 1.35 ml.
Assay of ATPase Activity
ATPase was determined
using a regenerating system for ATP as described by Pullman et al. (1960). One unit of ATPase activity is defined as 1 µmol of
ATP hydrolyzed per min. Specific activity is defined as units/milligram
of protein.
Protein Concentration
The concentration of
mitochondrial proteins was determined by a modified Biuret procedure
(Pullman et al., 1960) using bovine serum albumin as standard.
), 0.8-1 µmol of
ATP/min/mg, line 1, was in the range commonly observed with these
particles and a substantial rate of ATP synthesis with NADH as
substrate also was found (1.06 µmol of ATP formed/min/mg protein).
Washing with KCl increased the ATPase activity of the particles to a
value of 11 units/mg, line 2, but reduced the rate of ATP synthesis as
well as the P/O ratio. Reconstitution of KCl-washed particles with
F
, line 3, was without effect on ATPase activity but
substantially enhanced the rate of ATP synthesis. It should be noted
that ATPase activity of the particles in lines 1-3 of the table
was inhibited more than 90% by oligomycin.
Rate of Binding of ATP in High Affinity Catalytic Sites of
Reconstituted, KCl-washed ETPH(Mg
Binding
measurements were carried out using the techniques of acid quench and
cold chase as described previously (Penefsky, 1985a, 1985b). Fig.
1 A, lower curve, shows that relatively little
hydrolysis of 0.005 µ
M [)
-
P]ATP
(8%) occurred during 4 s of incubation with 0.125 mg of KCl-washed
ETPH(Mg
). However, the cold chase revealed that
substantial binding of [
-
P]ATP (50%) as
such occurred during the same time period, Fig. 1 A,
upper curve. The apparent rate of binding was similar to that
previously reported (Penefsky, 1985a), but reflected instead the lower
ligand concentrations used. Throughout this paper it was assumed that
each milligram of submitochondrial particles contained 0.4 nmol of
F
(Harris et al., 1977; Ferguson et al.,
1976; Beltran et al., 1986; Matsuno-Yagi and Hatefi, 1988).
Thus, the molar ratio of F
to ATP in the experiment was
calculated to be 10. Fig. 1 B shows that hydrolysis of
0.01 µ
M [
-
P]ATP in the acid
quench measurement, lower curve, was considerably enhanced in
the presence of 0.1 m
M ADP and that the acceleration of
hydrolysis in the cold chase, upper curve, was correspondingly
reduced. This observation is reminiscent of earlier findings that ADP
promoted the hydrolysis of ATP bound in high affinity catalytic sites
(Penefsky, 1985a). The rate of hydrolysis in the acid quench of 0.01
µ
M[
-
P]ATP in the presence of 1
m
M NADH, Fig. 1 C, lower curve, was
similar to that found in the absence of NADH, Fig. 1 A,
lower curve. The cold chase experiment, Fig. 1 C,
upper curve, indicates that a substantial amount of
[
-
P]ATP (23% of that which was added) was
bound in catalytic sites after 5 s of incubation in the presence of
NADH. However, in the presence of both ADP and NADH (Fig. 1 D),
that is, under the conditions of oxidative phosphorylation, almost no
ATP as such was found in the high affinity catalytic site. Hydrolysis
in the acid quench was 14% after 5 s while hydrolysis in the cold chase
was only 19%. The lower concentrations of enzyme and
[
-
P]ATP used in Fig. 1 A permitted better resolution of the initial portion of the
``on'' reaction. The results for Fig. 1,
A- D, were plotted in the form of a second-order rate
equation in Fig. 1 E. The calculated bimolecular rate constants
( k
), that is, the on rates, are summarized
in Table II. k
in the absence of any
additions, Fig. 1 A, 3.5
10
M
s
, compares well
with the value previously reported for KCl-washed submitochondrial
particles, Penefsky (1985a), of 4.0
10
M
s
. The small
differences in the values of k
for the
various experimental conditions of Fig. 1were reproducible
().
Rate of Binding of ATP in High Affinity Catalytic Sites
of ETPH(Mg
The measurements with
ETPH(Mg)
) were carried out under exactly the same
conditions used for the study of KCl-washed and reconstituted
submitochondrial particles except that the protein and
[
-
P]ATP concentrations were somewhat higher
in the former. The experiments of Fig. 2, ETPH(Mg
),
panel A, were done using an 80-fold excess of enzyme over
[
-
P]ATP, 2 mg of particles (0.8 nmol of
F
) and 0.01 nmol of [
-32P]ATP, may be
compared with those of Fig. 1 A, KCl-washed and
reconstituted particles. When the concentration of
[
-
P]ATP was increased 10-fold, the rate of
hydrolysis in the acid quench, panel B of Fig. 2,
lower curve, was considerably higher than that in panel A of Fig. 2, lower curve. Nevertheless, the on rate
constants were virtually identical in both experiments (Fig. 2,
legend). In addition, the cold chase showed that 37% of added
[
-
]ATP was bound as such (calculated from
the difference between cold chase and acid quench hydrolysis) in
catalytic sites of ETPH(Mg
) and activated particles
(Fig. 2, panel A and Fig. 1, panel A,
respectively) but only 24% in ETPH(Mg
) in panel B of Fig. 2. The presence of 100 µ
M ADP was
without effect on acid quench hydrolysis but reduced the level of bound
[
-
P]ATP as such to only 10% of that added,
Fig. 2 C. Similar low levels of
[
-
P]ATP binding were seen in the presence
of NADH, Fig. 2 D, and even less binding in the presence
of NADH and ADP, Fig. 2 E. P
was present in
all of these experiments but had little effect on either the on rates
or the ``off'' rates. Calculations of k
from the slopes of second-order plots, Fig. 2 F, were
carried out on the assumption that the particles contained 0.4 nmol of
F
/mg of protein and that the observed binding took place in
a single catalytic site. The results are shown in .
Similar to experiments with KCl-washed particles, small but
reproducible decreases in k
were observed
when the particles were energized (). Measurement of the Rate of Binding of
[
-
P]ATP in High Affinity Catalytic Sites of
ETPH(Mg
) Using Hexokinase-The rate of binding
of [
-
P]ATP was determined as
hexokinase-inaccessible ATP (see legend to Fig. 3) in order to
allow for the possibility that the presence of ADP, which is known to
inhibit ATPase activity (Pullman et al., 1960; Vasilyeva
et al., 1982; Feldman et al., 1985) would interfere
with measurements made via the cold chase technique.(
)
The on rate in the absence of ADP by the hexokinase method was 8
10
M
s
and may be compared with a value of 2.3
10
M
s
obtained by
the cold chase technique, Fig. 2 A. The higher value of the on
rate by the hexokinase method reflects the fact that 20-30% of
the bound radioactive ATP that is inaccessible to hexokinase, cannot be
chased into
P
. The on rate by the hexokinase
technique in the presence of 0.1 m
M ADP was 1.6
10
M
s
and
was the same in the presence of 1 m
M NADH.
Figure 2:
Rates of binding of
[-
P]ATP in catalytic sites of
ETPH(Mg
). The composition of the reaction mixtures
was exactly the same as described in Fig. 1 except that the
concentration of ETPH(Mg
) was 2 mg/ml. The
concentration of [
-
P]ATP was 0.01
µ
M for the experiments in panel A and 0.1
µ
M for the experiments in panels B-D.
Experiments in panels A and B were done in the
absence of NADH and ADP, in panel C, in the presence of 0.1
m
M ADP, in panel D, in the presence of 1 m
M NADH, and in panel E, in the presence of both NADH and
ADP. The designations in panel F on each of the slopes refer
to panels B-E. The calculated rate constant in the
absence of ADP and NADH was 2.3
10
M
s
; in the
presence of 0.1 m
M ADP, 1.6
10
M
s
; in the
presence of 1 m
M NADH, 1.0
10
M
s
and in the
presence of both ADP and NADH, 3.8
10
M
s
. The
calculated k
in panel A was 1.53
10
M
s
. The calculated rate constant in a different
preparation in the absence of ADP and NADH was 2.0
10
M
s
; in the
presence of 0.1 m
M ADP, 0.23
10
M
s
; in the
presence of 1 m
M NADH, 0.45
10
M
s
, and in the
presence of both ADP and NADH, 2.1
10
M
s
.
Figure 3:
Rate of binding of
[-
P]ATP in catalytic sites of
ETPH(Mg
) in the presence of ADP and NADH and in the
absence of P
in the buffer. Panel A, ATP binding
was measured as hexokinase-inaccessible ATP as described earlier
(Penefsky, 1985c). The reaction mixtures contained, in a final volume
of 1.0 ml, 2 mg of ETPH(Mg
), 20 m
M Tris-SO
, pH 8, 5 m
M MgSO
0.1
m
M ADP, and 0.25
M sucrose. The temperature was 23
°C. After a 1-min preincubation, the reaction was started by adding
1 m
M NADH followed 10 s later by 0.1 nmol of
[
-
P]ATP (specific activity 1.7
10
counts/min/nmol). Hexokinase (200 units) was then added
at the time points indicated on the abscissa, and the reaction
was continued for an additional 5 s before termination with 1.0 ml of 2
N HCl. The solutions were heated for 7 min in a boiling water
bath, and glucose-6-
P was separated and quantitated as
described under ``Experimental Procedures.''
Hexokinase-inaccessible ATP was calculated as the difference between
the amount of glucose-6-
P formed in reaction mixtures and
the amount formed in the absence of submitochondrial particles.
Panel B, the data of panel A are plotted in the form
of a second-order rate equation. k
was 5
10
M
s
Panel C, ATP binding was measured by cold chase as
described in Fig. 2. The composition of the reaction mixtures was
exactly as described for panel D of Fig. 2 except that P
was not present in the buffer. Hydrolysis during the acid quench
( lower curve) and cold chase ( upper curve) was
determined at points indicated on the abscissa. Panel
D, the data of panel C are plotted in the form of a
second-order rate equation. k
was 2.1
10
M
s
.
The on rate
constants in ETPH(Mg) in the presence of NADH and 0.1
m
M ADP were also measured under the exact conditions used in
but without P
(Fig. 3). This was done in order
to eliminate the possibility that ATP synthesis might interfere if it
occurred during the on rate measurements. k
in the absence of P
was 5.0
10
M
s
by the
hexokinase method and 2.1
10
M
s
by cold chase
(Fig. 3, panels B and D, respectively). In
reconstituted, KCl-washed ETPH(Mg
), the on rate in
the presence of NADH and 0.1 m
M ADP and in the absence of
P
was 1.0
10
M
s
. The Effect of ADP on the Energy-dependent Dissociation of
[
-
P]ATP from High Affinity Catalytic Sites
on ETPH(Mg
)-Incubation of submitochondrial
particles, containing [
-
P]ATP bound in high
affinity catalytic sites, under the conditions of oxidative
phosphorylation, that is, in the presence of NADH, ADP, and
P
, resulted in a kinetically competent, energy-dependent
dissociation of the bound radioactive nucleotide. That is, the rate of
dissociation of [
-
P]ATP was at least as
fast as the rate of ATP synthesis catalyzed by the same particles,
Penefsky (1985b). Fig. 4 B, upper curve shows that 10
µ
M ADP was sufficient to provide maximal dissociation of
bound [
-
P]ATP during a 2-s incubation with
NADH. In the presence of NADH and FCCP, Fig. 4 A,
lower curve, only about 4% of added
[
-
P]ATP was dissociated in the presence of
300 µ
M ADP. Two points of importance emerged from this
experiment. First, proton flux was itself sufficient to cause an
energy-dependent dissociation of bound ATP (Fig. 4 A, upper
curve (no added ADP) and Penefsky (1985b)). Second, ADP served, in
a cooperative manner, to enhance the energy-dependent dissociation of
bound [
-
P]ATP. In contrast to earlier
observations (Penefsky, 1985b), addition of NADH to submitochondrial
particles containing [
-
P]ATP bound in high
affinity catalytic sites did not result in an increase in hydrolysis of
[
-
P]ATP.
NADH used in the
earlier studies apparently contained a contaminant responsible for the
stimulation of hydrolysis. The Rate of Dissociation of [
-
P]ATP Bound in
High Affinity Catalytic Sites-The rate of dissociation of bound
[
-
P]ATP in the presence of NADH or NADH and
ADP was too fast to be measured by manual methods. Fig. 5 shows the
results of a series of experiments with a rapid mixing device described
earlier (Grubmeyer et al., 1982). The enzyme-substrate complex
was formed in the first section of aging hose and was then mixed in a
second section of aging hose with NADH and hexokinase or NADH, 0.1
m
M ADP, and hexokinase. The time of incubation in the
energized state is shown on the abscissa and was determined by
a delay programmed into the instrument. It was necessary to use very
large amounts of hexokinase (800 units/ml) in order to compensate for
the relatively slow rate of the hexokinase reaction. Nevertheless, it
was not possible to use higher concentrations of ADP or temperatures
greater than 23 °C because the resulting higher rates of
dissociation would not have been resolved by the rate-limiting
hexokinase reaction. The Rate of Dissociation of [
-
P]ATP Bound in
High Affinity Catalytic Sites of F
-reconstituted,
KCl-washed ETPH(Mg
)- shows that
the rate of dissociation of bound ATP in the absence of any additions
was 10
s
, in agreement with
earlier values for the soluble, Grubmeyer, et al. (1982) and
the membrane-bound, Penefsky (1985a), forms of the ATPase. The presence
of 100 µ
M ADP in the reaction mixture was without effect
on the rate of dissociation. However, in the presence of NADH or NADH
and ADP, the rate of dissociation was 0.083 and 0.24
s
, respectively. Thus, under the conditions of
oxidative phosphorylation, that is, in the presence of NADH, ADP, and
P
, the rate of dissociation of
[
-
P]ATP bound in high affinity catalytic
sites was increased about 10
times above the non-energized
rate.
Figure 4:
Effect of ADP on the dissociation of ATP
bound in high affinity catalytic sites of energized and nonenergized
ETPH(Mg). A, 2 mg of
ETPH(Mg
) was preincubated for 1 min at room
temperature in a buffer containing 20 m
M Tris-SO
,
pH 8, 5 m
M KH
PO
, pH 8, 5 m
M MgSO
, 50 m
M glucose, and 0.25
M sucrose. The final volume was 1.0 ml. After incubation with 0.1
nmol of [
-
P]ATP for 10 s, 200 units of
hexokinase were added to convert any free
[
-
P]ATP to glucose-6-
P. Five s
later, the indicated amounts of NADH or NADH and ADP were added. Where
indicated (+ FCCP), 20 µ
M FCCP dissolved in
ethanol was added to a reaction mixture identical with that described
above. All incubations were continued for 2 s after the addition of
NADH or NADH and ADP, and the reactions were terminated by adding 1 ml
of 2
N HCl. The amounts of glucose-6-
P formed
were determined as described under ``Experimental
Procedures.'' In a separate experiment, 200 units of hexokinase
converted 0.1 nmol of [
-
P]ATP to
glucose-6-
P in less than 5 s. B, the reactions
were carried out exactly as described for panel A. ADP was
added to a final concentration indicated on the abscissa with
1 m
M NADH. The amounts of glucose-6-
P formed
after 2 s were determined as described under ``Experimental
Procedures.'' The total amounts of
[
-
P]ATP free (set equal to the amount of
glucose-6-
P formed) are shown on the
ordinate.
The rate of the energy-dependent dissociation of
[-
P]ATP ( k
) as
well as the rate of binding of [
-
P]ATP
( k
) was the same in the presence and absence
of added P
.
It is interesting that energization
of Escherichia coli membrane vesicles decreased the
dissociation constant for P
( K
P
)
and blocked rebinding of ATP (Al-Shawi et al., 1990; Weber
et al., 1994).
, were calculated from the
ratios of the individual rate constants,
k
/ k
(). Energization decreased the affinity for
[
-
P]ATP in high affinity catalytic sites by
five to six orders of magnitude. Calculation of
G
from the expression
G
=
- RT ln K
provided the change in standard free energy, in kcal/mol,
equivalent to the individual dissociation constants. The observed free
energy change (
G) was calculated from the expression
G = -RT ln
K
. It may be
seen that energization with NADH, 0.1 m
M ADP, and P
gave
G of 8.5 kcal/mol (). The Rate of Dissociation of [
-
P]ATP Bound in
High Affinity Catalytic Sites of
ETPH(Mg
)-Similar to KCl-washed particles, the
off rates for [
-
P]ATP were accelerated by
about five orders of magnitude in the presence of NADH and by about six
orders of magnitude in the presence of NADH and 0.1 m
M ADP
(). Calculation of K
from the ratio of the reverse and forward rate constants
indicated that the affinity for ATP was reduced six orders of magnitude
in the presence of NADH and seven orders of magnitude in the presence
of NADH and ADP. Thus, the observed free energy change
(
G) in the transition from non-energized to energized
states was 8 kcal/mol in the presence of NADH and 9.7 kcal/mol in the
presence of NADH and ADP ().
Effect of ADP Concentration and Temperature on the Rate
of ATP Synthesis by ETPH(Mg
The turnover number for
ATP synthesis in both types of particles was sensitive to ADP
concentration and temperature, I. Maximal rates of ATP
synthesis were observed in both types of particles at 1 m
M ADP
and 30 °C. The off rate was commensurate with the turnover number
of the enzyme under the same conditions described for the off rate
measurement, that is, 0.1 m
M ADP and 23 °C (Tables II and
III).
) and Reconstituted,
KCl-washed ETPH(Mg
)
F
-ATPase of beef heart
mitochondria for product ATP during oxidative phosphorylation. It is
shown that the affinity of the non-energized enzyme for ATP is high in
both ATPase-activated and non-activated submitochondrial particles. The
dissociation constant for ATP
( K
) was calculated from the
ratio of reverse off ( k
) and forward on
( k
) rate constants. In non-energized
membranes, K
was
10
M ( and Penefsky, 1985a).
However, in the presence of NADH, 0.1 m
M ADP and
P
, K
was about
60 µ
M (), ETPH(Mg
). Thus,
the affinity of the enzyme for ATP must decrease seven orders of
magnitude when the proton pumps are activated by initiating
respiration. The observed change in
K
is expressed almost
entirely as an increase in the rate of ATP dissociation
( k
increased by five orders of magnitude
over the rate observed under non-energized conditions ())
with only small changes in the rate of ATP binding to catalytic sites
under energized conditions ().
was enhanced in the presence of ADP. The
enhancement was dependent on the ADP concentration and was seven times
greater in the presence of ADP than in its absence (Table II). The
ADP-dependent rate enhancement did moreover reflect an energy-dependent
cooperativity between catalytic subunits since FCCP prevented the
ADP-promoted dissociation of bound ATP, Fig. 4. Thus, the energy
of respiration brought about cooperativity between subunits of the
enzyme with respect to release of product ATP. These observations are
compatible with earlier findings of cooperativity with regard to both
ATP hydrolysis (Grubmeyer and Penefsky, 1981a, 1981b) and synthesis
(Matsuno-Yagi and Hatefi, 1985). The observed free energy change
associated with the cooperative effects of ADP was calculated to be 1.7
kcal/mol (). This cooperative contribution to net ATP
synthesis is of considerable importance since the rate of ATP synthesis
at low concentrations of ADP is very low (Matsuno-Yagi and Hatefi,
1985; Perez and Ferguson, 1990a, 1990b). Further support for the
importance of ADP concentration is shown in Fig. 2and
, ETPH(Mg
). In the presence of NADH (but
absence of added ADP), K
was about 3 µ
M. Thus the affinity for product ATP in
the presence of low ADP levels was too high to permit efficient net ATP
synthesis. These observations are consistent with conditions observed
during respiratory control. Low rates of ATP synthesis and of
respiration occur at low concentrations of ADP.
. Thus, the standard
free energy associated with a K
about 10
M was 15-17 kcal/mol
(). The observed free energy change (
G) on
the transition from non-energized membranes to membranes energized by
NADH alone was 7.5 kcal/mol (reconstituted, KCl-washed particles) and 8
kcal/mol (ETPH(Mg
)) (Table II). It is significant
that proton flux was itself sufficient to bring about a change in the
affinity of the enzyme for ATP of six orders of magnitude. These values
of 7.5-8 kcal/mol are the most reasonable values to compare with
reported values for the standard free energy of hydrolysis of ATP under
physiological conditions of -8.4 kcal/mol (Rosing and Slater,
1971). In the presence of NADH, 0.1 m
M ADP and P
(that is, under the conditions of oxidative phosphorylation) a
further increase in
G was observed, 8.5 and 9.7 kcal/mol
for KCl-washed ETPH(Mg
) and
ETPH(Mg
) particles, respectively (). It
should be emphasized that these values of
G were obtained
from experiments with suboptimal concentrations of ADP. Maximal rates
of ATP synthesis (1 µmol of ATP/min/mg) were observed with
ETPH(Mg
) particles at 1 m
M ADP
(I). The calculated turnover number for this rate is 44
mol of ATP/mol of F
per s (I). If the turnover
number was set equal to the off rate, k
,
the calculated K
at 30
°C in the presence of 1 m
M ADP would be in the m
M range (consistent with the m
M range of ATP concentration
in the mitochondrial matrix) and
G would be
correspondingly larger. It was not possible to measure the rate of ATP
dissociation directly, as described in Fig. 4, in the presence of
1 m
M ADP or at higher temperatures, because the
hexokinase-catalyzed conversion of ATP to glucose-6-P was too slow to
resolve the kinetics. On the other hand, measurement of the rate of
dissociation in the presence of 10 µ
M ADP and
ETPH(Mg
) yielded a value intermediate between 0.32
and 2.3 s
. The temperature dependence of ATP
synthesis in I can be explained as an effect of
temperature on the rate of respiration.
and their implications
for
G may be compared with earlier reports of
K
in light-energized
spinach chloroplasts of 7 µ
M (Magnusson and McCarty, 1976)
and of 16 µ
M with phosphorylating vesicles from P.
denitrificans by Perez and Ferguson (1990a, 1990b). However, the
chloroplast values were obtained by measuring the exchange of ATP
between the enzyme and the medium in the presumed absence of ADP.
Consequently, the 7 µ
M K
of Magnusson and
McCarty (1976) might best be compared with a
K
of 0.1-3 µ
M observed with submitochondrial particles in the presence of NADH
alone (). Perez and Ferguson (1990a, 1990b) determined
K
, which they could not
measure directly in their system, by equating it with a measurable
product inhibition constant,
K
. It is more difficult to
compare this latter value with the numbers reported in this paper, but
if product inhibition by ATP may be presumed to reflect ATP binding to
an enzyme which does not contain ADP in a second catalytic site, then
the 16 µ
M value of Perez and Ferguson may also be
comparable with numbers observed in this paper in the absence of added
ADP, that is 0.1 to 3 µ
M. It is important to point out
that in the case of maximally phosphorylating submitochondrial
particles exhibiting a K
in
the millimolar range, it is not necessary to postulate product
inhibition by ATP (Perez and Ferguson, 1990a, 1990b) in order to
understand how net formation of ATP could occur in the presence of
m
M concentrations of intramitochondrial ATP.
) under unisite
conditions was slow (10
s
) and
due to the slow rate of dissociation of P
from
membrane-bound F
(Souid and Penefsky
; Penefsky,
1985a). Matsuno-Yagi and Hatefi (1993) found a P
off rate
of 0.12 s
using conditions apparently the same as
those of Penefsky (1985a). An explanation of the discrepancy is not
apparent. The fact that more [
-
P]ATP was
available for energy-dependent dissociation from KCl-washed particles
than from ETPH(Mg
) was explained by the slower rate
of unisite hydrolysis of ATP by the former particles. A slow rate of
hydrolysis of ATP, consistent with unisite catalysis, was observed in
ETPH(Mg
) when the ratio of F
to
[
-
P]ATP was increased to 80-fold ( panel
A of Fig. 2). Thus, it is possible that the hydrolysis in
panel B of Fig. 2( lower curve), that is, at a
ratio of F
/[
-
P]ATP of 8-old,
reflects catalysis at more than one site. Nevertheless, the on rate was
the same in both experiments (legend of Fig. 2). This is due to
the fact that the rate of ATP binding at a second catalytic site is the
same as the rate of binding at the first site (Cross et al.,
1982).
) or 2 mg of
ETPH(Mg
) was fully capable of binding 97% or more of
0.1 nmol of [
-
P]ATP added to reaction
mixtures (measured as hexokinase-inaccessible
[
-
P]ATP).
) are
approximately 10-fold lower than for reconstituted, KCl-washed
ETPH(Mg
). There is an ambiguity in the calculation of
the on rate since it requires a value for the concentration of
membrane-bound F
. Throughout this study, we have used a
value of 0.4 nmol of F
/mg of particle protein (Harris
et al., 1977; Ferguson et al., 1976; Beltran et
al., 1986; Matsuno-Yagi and Hatefi, 1988). This value seems
reasonable for ATPase-activated particles, particularly reconstituted,
KCl-washed ETPH(Mg
). However, if one assumes that the
concentration of available catalytic sites on
ETPH(Mg
) is about one-tenth that of KCl-washed
particles (commensurate with the difference in ATPase activities
()), then the calculated on rates for
ETPH(Mg
) would rise about 10-fold.
M
s
). If it did
occur, such binding did not seem to have a noticeable effect on the
reduced rate of ATP binding under energized conditions ().
In addition, it may be noted that in experiments with
ETPH(Mg
) all of the
[
-
P]ATP bound to the membranes (determined
as the difference between cold chase and acid quench hydrolysis) was
dissociated within 1 s after adding NADH and 0.1 m
M ADP.
This experiment argues strongly for homogeneity in the high
affinity catalytic sites that bind [
-
P]ATP.
In non-energized ETPH(Mg
), 2 mg of the particles bind
about 97% of the added 0.1 nmol [
-
P]ATP in
5 s. About two-thirds of that [
-
P]ATP is
bound in the catalytic sites of the enzyme and one-third is not
accessible to hexokinase and cannot be promoted to hydrolyses by cold
chase or to dissociation by NADH or NADH and ADP.
In 5 s,
one-third of the total [
-
P]ATP is
hydrolyzed (Fig. 2, panel B, lower curve), and
one-third can be promoted to hydrolysis by cold chase (Fig. 2,
panel B, upper curve), ADP, or NADH and ADP in the
presence of FCCP.
In the absence of FCCP, NADH or NADH and
ADP promoted the release of [
-
P]ATP that
was bound as such rather than its hydrolysis.
There was no
detectable cold chase [
-
P]ATP following
energy-dependent dissociation.
These results support the
conclusions that the cold chase [
-
P]ATP is
the same as NADH-dissociable [
-
P]ATP and
the same catalytic sites participate in the on rate and off rate
measurements.
. For
example, the on rate in non-energized ETPH(Mg
) was
only 8-10-fold lower than the rates reported for ATPase-activated
particles (Penefsky, 1985a) and soluble F
(Grubmeyer et
al., 1982). In addition, the decrease in the on rate values
observed in the presence of NADH, ADP, or NADH and ADP was very similar
in both types of particles. The on rate values measured with cold chase
or hexokinase trap were similar. Somewhat higher numbers obtained with
hexokinase reflect the fact that 20-30% of bound ATP, that is,
ATP inaccessible to hexokinase, could not be chased into P
or dissociated by NADH oxidation. The amount of chaseable
[
-
P]ATP bound to ETPH(Mg
)
was equal to the amount of [
-
P]ATP
dissociated by NADH. This observation supports the conclusion that
[
-
P]ATP was bound to catalytic sites of
coupled F
F
complexes during on rate
measurements. Two further indications that the reported values for the
ATP on rate are reasonable is found in the fact that, first, the
K
values calculated from
the ratio of the rate constants are in good agreement with values
reported by Magnuson and McCarty (1976) and by Perez and Ferguson
(1990) and second, that the decrease in the on rate for ATP in the
presence of NADH and ADP is consistent with the observed increase in
the on rate for ADP during ATP synthesis (Matsuno-Yagi and Hatefi,
1986). Finally, in spite of the insensitivity of the thermodynamic
calculations to differences in
K
,
G should
not exceed the phosphorylation potential,
G
,
that is, 10.6 kcal/mol (Ferguson and Sorgato, 1977). Thus, during
steady state ATP synthesis,
G in
ETPH(Mg
) was about 4.8 kcal/mol (15.4 kcal/mol minus
10.6 kcal/mol, ). If the off rate under these conditions
is taken to be 44/s (I),
K
would be 0.3 m
M and k
1.5
10
M
s
. These
considerations provide insight into observations that ATP synthesis can
occur readily even at high ATP/ADP ratios (Ferguson and Sorgato, 1977).
This is so because under energized conditions the affinity of catalytic
sites for ATP was reduced more than seven orders of magnitude
(), while the affinity for ADP increased during ATP
synthesis (Matsuno-Yagi and Hatefi, 1986).
-fold decrease in affinity. Second, the
cooperative effects of ADP which, at 0.1 m
M concentrations,
decreased the affinity an additional 10-fold. The implications of these
calculations is that under the conditions of oxidative phosphorylation
the dissociation constant for ATP is in the m
M range.
Table:
ATP hydrolysis and synthesis by submitochondrial
particles
), reconstituted, KCl-washed
ETPH(Mg
) and methods for measurement of ATPase,
respiration, and phosphorylation are described under
``Experimental Procedures.'' The numbers in parentheses refer
to rates of ATP hydrolysis in the presence of 5 µg of oligomycin.
All of these experiments were carried out at 30 °C.
Table:
Rate constants, equilibrium constants and free
energy changes during ATP synthesis catalyzed by
ETPH(Mg) and KCl-washed ETPH(Mg
)
G (the observed free energy change)
was calculated from the expression
G =
- RT ln
K
.
Table:
Effect of ADP concentration and temperature on
the rate of ATP synthesis by ETPH(Mg) and
reconstituted, KCl-washed ETPH(Mg
)
,
membrane-imbedded portion, and F
, extrinsic, easily
solubilized portion of the F
F
-ATPase; FCCP,
carbonyl cyanide p-trifluoromethyoxyphenylhydrazone; TNP,
trinitrophenyl; OSCP, oligomycin sensitivity-conferring protein.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.