ATP-hydrolysis in chloroplasts: evidence for the participation of three ATP binding sites

288 Biochimica et Biophysica Acta, 1141 (1993) 288-296 © 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2728/93/$06.00

BBABIO 43768

ATP-hydrolysis in chloroplasts: evidence for the participation of three ATP binding sites

Andreas Labahn and Peter Gr~iber Biologisches Institut, Universith't Stuttgart, Stuttgart (Germany)

(Received 24 June 1992)

Key words: Chloroplast; ATP hydrolysis; ATPase, H +-; FoFz; Binding site

The H+-ATPase from chloroplasts, CFoF ~, is reduced by illumination of thylakoids in the presence of thioredoxin and dithiothreitol. The endogeneous nucleotides are removed by a washing procedure so that the inactive, reduced enzyme contains one tightly bound ATP per CFoF I. Upon activation of the enzyme by a ApH/A~b jump this ATP is not released. The inactivation of the enzyme occurs with first order kinetics with a half-lifetime of 450 s (rate constant kin = 1.5 • 10 -3 S- l) without rebinding of ADP. Obviously, there exist at least two different inactive states of the enzyme, one with a tightly bound ADP and one without bound ADP. The maximal turnover of the enzyme is 30 s- ~ before and after washing. The rate of ATP hydrolysis was measured from 1 nM to 1 mM ATP with this enzyme. A sigmoidal dependence of the rate is observed at low ATP concentrations, reaching a plateau rate of 0.007 s- ~ between 25 nM and 55 nM, and the half-maximal rate is reached at 15 nM. From the supralinear increase of the rate below 25 nM it is concluded that two catalytic sites are involved in this range. Above 1 ~M ATP the kinetics of ATP-hydrolysis can be described by Michaelis-Menten kinetics with Vmax = 30 s-~ and K~ = 220/~M. The results indicate the participation of at least three different sites in ATP-hydrolysis catalyzed by the membrane-bound CFoF v

Introduction

Membrane-bound H÷-ATPases ( 'FoF1-ATPases ') catalyze reversibly ATP synthesis/hydrolysis coupled with a t ransmembrane proton transport in bacteria, chloroplasts and mitochondria [1-3]. The enzyme has a hydrophilic F 1 part which contains the six nucleotide binding sites [4-6] and a hydrophobic F o part which is inserted into the membrane and is supposed to act as a proton channel. Three of the nucleotide binding sites are located on the/3-subunits (perhaps on the interface between a- and /3-subunits) and these have catalytic propert ies [7-10].

For the isolated F 1 part the rate of ATP hydrolysis was measured as a function of the ATP concentration. These measurements indicate the existence of 2 - 4 catalytic sites in mitochondrial F~ [11-13] and two sites [14] or three sites [42] in the F 1 part from chloroplasts, CF v However, the relevance of these data - especially those from CF~ - to the mechanism of proton transport

Correspondence to: P. Gr~iber, Biologisches Institut, Universit~it Stuttgart, Pfaffenwaldring 57, D-7000 Stuttgart 80, Germany. Abbreviations: CFoFt, H+-transloeating ATPase from chloroplasts; CF1, hydrophilic part of the H +-ATPase from chloroplasts; Pi, or- thophosphate.

coupled ATP hydrolysis is not clear. (1) ATP-hydrolysis is not coupled with proton translocation. (2) The isolated CF~ requires special t reatments in order to catalyze ATP hydrolysis. After these treatments, the enzyme requires Ca 2÷ as cofactor instead of Mg 2÷. The CaE÷-ATPase activity shows two apparent Michaelis- Menten constants ( K ~ - - 4 0 nM and K ~ = 200 /xM [141).

When MgZ+-ATPase activity is measured with the m e m b r a n e - b o u n d H÷-ATPase from chloroplasts, CFoF 1, only one apparent K ~ between 70 and 150 /zM was found [15-19]. However, the lowest ATP concentration used was about 3 ~ M and from the data with F t from mitochondria and E. coli it may be expected that ATP hydrolysis catalyzed by other sites occurs at substrate concentrations lower than the enzyme concentration.

Cunningham and Cross [13] showed with mitochondrial F~ that one catalytic site is operating only if the enzyme-to-substrate ratio is greater than 10: 1. An increase of the ATP concentration to stoichiometric amounts led to a significant occupation of a second site, as observed by the increase in the dissociation constants for Pi and ADP. AI-Shawi et al. [20] measured with F~ from E. coli the rate constant for ATP binding at different enzyme/ subs t r a t e ratios. In this case, the rate constant for ATP binding was acceler-

ated when the enzyme-to-substrate ratio was equal to 1.

In this work, we have measured the rate of ATP hydrolysis from nanomolar to millimolar ATP concentrations catalyzed by CFoF 1 bound at thylakoid membranes. In thylakoid membranes CFoF 1 contains 2 ATP and 1 ADP per CFoF l and ADP is released upon activation of the enzyme [21-23]. In the presence of Pi, the released ADP is phosphorylated to ATP and therefore, free ATP is always present with the same concentration as the enzyme [24]. In order to obtain a substoichiometric substrate-to-enzyme ratio, the endogenous nucleotides must be removed by a washing procedure while the enzyme activity remains constant. We de- scribe a thylakoid preparation which fulfills these re- quirements and all the following results are obtained with these 'washed thylakoids'.

Materials and Methods

Preparation of thylakoids with low nucleotide content ('washed thylakoids')

Class II chloroplasts were isolated as described in Ref. 25, resuspended in buffer 1 (20 mM Tricine-Na (pH 7.4), 100 mM sorbitol, 25 mM NaCI, 2 mM MgCI 2, 30% (v/v) ethylene glycol), chlorophyll concentration 2 mM and rapidly frozen and stored under liquid nitro- gen. Before the washing procedure, ethylene glycol was removed by dilution of 500 tzl thylakoid suspension with 7 ml buffer 2 (5 mM Tricine-Na (pH 8.0), 50 mM sorbitol, 2 mM MgCI2). The solution was centrifuged 5 min at 2800 × g and the pellet was resuspended in 200 ~1 buffer 2.

In order to activate and reduce the enzyme, 75 tzl thylakoids were added to 425 /~1 buffer 3 (5.9 mM Tricine-Na (pH 8.05), 0.6 mM EDTA, 2.95 mM MgCI 2, 3.5 /zM thioredoxin, 23.5 tzM pyocyanin, 1.2 mM dithiothreitol) and illuminated at room temperature with saturating white light. After 1 min, 1 ml buffer 2 was added and the suspension was centrifuged 10 s at 10000 × g (Beckman microfuge E equipped with a perspex lid) under continuous illumination with a pro- jector lamp (250 W). Then, the pellet was resuspended in 1.5 ml buffer 2 and centrifuged again as described above. The pellet was resuspended in 400/~1 buffer 3 (final chlorophyll concentration about 100 ~M). The

red CFoF ~ was now in the active, reduced state (E a ). The thylakoids were stored in the dark on ice. 30 rain later

(E i ). Washed the enzyme was completely inactivated red thylakoids, which can be kept on ice for about 4 h, show - after reactivation - the same rate of ATP-hydrolysis as before the washing procedure.

Nucleotide content In order to characterize the washed thylakoids, the

nucleotide content was determined by using the lucifer-

289

ine/luciferase technique [24,26] and compared with the untreated thylakoids. A ratio of 730 chlorophyll per CF 0 F x was measured by immune electrophoresis [27,28]. The fraction of active enzymes was determined by measuring the maximal rate of ATP hydrolysis at the initial concentration of 1 mM ATP for every set of experiments.

A TP-hydrolysis The washed thylakoids were reactivated by a

ApH/Ad: jump as follows: Thylakoids with CFoF 1 in the state E[ ed (40 ~1) were incubated 30 s with 100/~1 of an acidic buffer 4 (40 mM succinate-Na (pH 5.5), 2 mM MgC12, 1.2 mM KCI, 2 mM NaHzPO 4, freshly added 20 /xM DCMU (3-(3,4-dichlorophenyl)-l,1- dimethylurea) and 5/zM valinomycin). Then, 150/zl of buffer 5 (200 mM Tricine-Na (pH 8.3), 120 mM KOH, 2 mM MgCI 2, 2 mM NaH2PO 4, freshly added 10/~M DCMU) was added inducing a ApH/A~b jump (ApH 2.2, A¢ 90 mV). After 15 s, 250 ~I of this solution was mixed with 50 /zl buffer 6 (100 mM Tricine-Na (pH 8.3), 60 mM KOH, 2 mM MgC12, 2 mM NaH2PO 4, freshly added 10/xM DCMU, 36 mM NH4CI as uncoupler, [y-32p]ATP (2 kBq, Amersham-Buchler) and unlabelled MgATP between 6 nM and 6 mM. The mix- ture of all these solutions is hereafter termed 'reaction medium'. The initial ATP concentration in each experiment, ATP °, is the sum of added and endogenous free ATP. The endogenous free ATP was determined by adding 100 /xl of the thylakoid suspension after the ApH/A~b jump to a luciferin/luciferase assay.

Every 10 s, up to a total reaction time of about 50 s, 50 txl samples were taken and denatured by 50 /~I trichloroacetic acid (final concentration 2% (w/w)). Then, 1 mM unlabelled Mg-ATP was added to each sample.

[32p]pi was extracted from the denatured samples as phosphomolybdate complex as described earlier [29]. With this method the sum of enzyme-bound phosphate (Pib) and released phosphate (Pif) was determined. In order to measure the free phosphate released from the enzyme, the reaction medium was not denatured. The samples (100 ~1) were taken at the appropriate reaction time by a 1 ml syringe and immediately pressed through a filter (No. 8110, Tecnomara). The separation time was approx. 2 s. The radioactive Pi in the filtrate was measured as described above.

The rates are based on the enzyme concentration, they are expressed in M ATP/(M CFoF 1 s), i.e., in s-i.

Results

Nucleotide content of the washed thylakoids In order to reduce the nucleotide content, the thyl-

akoid membranes were washed as described in Materi- als and Methods. The free and the bound nucleotides

290

TABLE I

The effect of washing on free (f) and bound (b) nucleotides

The free and bound nucleotides were measured before the washing procedure (E °x and E~d), after washing (E~ ca) and after washing and activation ~cd (E a ) as described in Refs. 24 and 26. The free nucleotide concentrations are the absolute concentrations of the different steps of the preparation. The last column indicates the conditions when ATP hydrolysis is started by addition of ADP. In this case the enzyme concentration was 20 nM.

Nucleotides Before After After washing washing washing and activation

E oX E~cd E~ cd E.~ ~d

[ADP]f / [CFoF I ] 0.26 0.10 0.08+0.05 0.15_+0.1 [ADP] f /nM 133 2 14 + 9 3 + 2 [ATPIf / [CFoF 11 0.02 1.13 0.0 +0.1 0.1 -+0.1 [ATP] f /nM 10.2 22.6 < 0.1 2 -+ 2 [ADP]b/[CFoFII 0.94 0.0 0.05+0.05 0.0 -+0.05 [ATP]b/[CFoF ~] 1.89 1.89 1.7 _+0.2 1.2 _+0.2

were measured before and after washing. Table I shows the result: before washing, the enzyme contains about 2 ATP and 1 ADP per CFoF ~ and after washing and activation, 1 ATP. The conditions listed under Er~ d after washing represent the initial state of the enzyme with respect to free and bound nucleotides for all further experiments. The endogenous free ADP and free ATP concentration are 3 nM and 2 nM, respectively, at a CFoF ~ concentration of 20 nM. The conditions listed under E red before washing and activation represent the initial state of the enzyme in earlier work [24]. Several other washing procedures (higher dilution, without illumination, several centrifugations, etc.) were tested. However, in all cases the amount of bound nucleotides a n d / o r the concentration of free nucleotides were higher.

During the washing procedure the enzyme might be partially denatured. Therefore, the maximal rate of ATP-hydrolysis, Vmax, was measured after washing. 15 s after the reactivation, 1 mM ATP and uncoupler were added and the rate of ATP hydrolysis was measured as described in Materials and Methods. A rate of (30 5: 5) s-~ was obtained. As a control, the rate before the washing procedure was determined. In this case, the inactive, oxidized enzyme was reduced and activated by illumination for 1 min in the presence of dithiothreitol and thioredoxin. 15 s after reactivation with a ApH/A~b jump, ATP and uncoupler were added. It resulted in a rate of (28 5: 4) s - t . Obviously, no denaturation oc- curred during the washing procedure. Surprisingly, these rates are much lower than those reported earlier under similar conditions (100 s - ] ) [19,30]. The only obvious difference was the presence of LiCI in our earlier measurements. (The LiCI arises from the preparation of [y- 32p]ATP.) Therefore, ATP-hydrolysis was

measured in the presence and absence of 150 mM LiCI. Fig. 1 (top) shows the P~ formed as a function of reaction time, indicating a maximal turnover of 79 s- ( + LiCI) and 28 s - ] ( - L i C I ) in the presence of 1 mM ATP. Fig. 1 (bottom) shows the same type of experiment under uni-site conditions, i.e., in the presence of 5 nM ATP. The turnover rate in this case is 1.4.10 - 3

s - t (+LiCI) and 0 .4 .10 - 3 s - ] ( - LiCI). In both cases, the rates differ by a factor of 3. The control values at t = 0 represent t h e [32p]p i in the [y-32p]ATP, which are the same in the presence and absence of LiCI (see Fig. 1). This indicates that LiCI does not influence P~ complexation and extraction of the phosphomolybdate complex, but influences the enzyme-catalyzed ATP hydrolysis. Since the rate is changed by the same factor in the concentration range betweeen nano- and millimolar the relative rates (U/Umax) do not depend on the LiCI concentration. Additionally, Fig. 1 bottom shows two controls. In one control, no enzyme was added to the reaction medium while in the second one, denatured enzyme was added. In both cases, the control values (i.e., the [3ep]p i in the [y-32p]ATP remained constant, whereas in the presence of thylakoids a small but statistically significant increase of [32P]P i is observed.

80 ~L

v 60 "1o Q)

E 4 0 o

2O CL--

[A~p]O-1 mM • 150 mM UCl [CFoF1]O-11 nM o without UCl

• ~ MATP M c r,F,..

28 M An = . CFoF, .o

0 i ! i i i

0 20 40 60

t i m e , tx ( s )

.--, 1000 [ATP] 0 " 5 nM • 150 mM LiCI ~E

[CFoF1] O- 11 nM o without UCI • ¢ k

.lo 7 5 0 mMATP ® . % r I ., E

• 0 . 4 m id A I ~

a.- 250 ~ U C~o~.,

T Control 0 i i i i i

0 10 20 30 40 50

time, tx(s) Fig. 1. Effect of LiCI on the rate of ATP-hydrolysis catalyzed by the active, reduced enzyme. Top: in the presence of 1 mM ATP; bottom: in the presence of 5 nM ATP. Controls: 5 nM [7-32p]ATP is incubated in the reaction medium in the presence of denatured enzyme (open squares) or in the absence of enzyme (filled squares).

° ~

t l .

t ( s )

3 0

165

_~ ~ 255 15

I I

10 20 30 40 5 0

time, t x ( • )

>]>o 0 . 0 ~

c • - - - 450 a " -0.5

-1.0

Tt/2

k in - 1 . 5 . 1 0 - 3 a -1

i i I I i

0 60 120 180 240 300 time, t ( s )

Fig. 2. Inactivation of the active, reduced enzyme. The enzyme was activated with ApH/A¢ jump, [CFoF1] *= 19 nM, [ATP]*= 1 mM. Top: Time-course of Pi formation at different times after the activation. The numbers at the slopes give the rate in M ATP/(M CFoF ~ s). Bottom: Logarithmic plot of the relative rate of ATP-hydrolysis v / v o vs. time after the zlpH/A4, jump. Rates are calculated from the data at the top and additional measurements. Initial rate (15 s

after the ApH/A~ jump) v 0 = 28 M ATP/(M CFoF 1 s).

The active, reduced enzyme, F red which catalyzes ATP hydrolysis, is a metastable state of the enzyme. When the membrane energisation is dissipated, the enzyme returns after rebinding of ADP to its stable, inactive state.

In the washing procedure, the free and the bound ADP are drastically decreased. In order to determine the time range in which the state of the enzyme remains unchanged, the inactivation of W ed was mea- - - a

sured. After the washing procedure, the enzyme was activated by a A p H / A ~ jump as described in Materi- als and Methods and stored at 20°C.

At different times, ATP hydrolysis was started by the addition of 1 mM ATP and uncoupler and the amount of Pi formed was determined. Fig. 2 top shows the result at 30 s, 165 s and 255 s after the activation. From the slopes of these curves the rates v are calculated and the relative rates V/Vm~ x (Vma x, maximal rate of ATP hydrolysis measured 15 s after the ApH jump) then give directly the fraction of active enzymes. Fig. 2 bottom shows a logarithmic plot of the relative rates as a function of time after activation. The inactivation can be described by a first-order reaction with a half-lifetime of 450 s. This corresponds to a rate constant of 1.5" 10 -3 S - I .

291

The following experiments are all carried out in the time range up to maximally 60 s after activation. Ac- cording to the data from Fig. 2, within this time range only 9% of the enzymes are inactivated. Such an effect is neglected in further evaluations. Fig. 3 shows ATP hydrolysis at different substrate concentrations, indicating a linear increase of Pi formed with reaction time. The curves are shifted arbitrarily along the ordinate for a clearer presentation. The slopes of these curves represent the rates of ATP hydrolysis. From the data in Fig. 3 and additional measurements, the rates of ATP hydrolysis were calculated and plotted in Fig. 4.

Fig. 4A shows the rates up to 7 nM ATP where a linear increase of the rate with increasing ATP concentration is found. The slope is k ' - - 1.5 • 105 M-1 s-l. Above 7 nM the rate increases supralinearly and reaches a plateau between 25 nM and 55 nM (Fig. 4B and 4C). In the range between 7 nM and 25 nM the curve has a nearly constant slope, k " = 3 - 1 0 5 M -1 s-I. In the micromolar range (Fig. 4D) again a linear increase is found with the slope k ' = 3.105 M-~ s-1, and finally in the millimolar range, saturation is observed (Fig. 4E). The data in the millimolar range can be described by Michaelis-Menten kinetics and a least-squares fit of the data gives v ~ = 30 s- i and K~ = 220/zM. This functional dependence is indicated by the solid line in Fig. 4E. Since the supralinear increase of the rate in the nanomolar range requires the participation of two catalytic sites, we have, to-

E k_ 0

[CF 0 F 1 ]0=20 .M [ATP ] 0 ~ ~ _ 103 35 nM

~ 18 nM

• ~ 13nM =r

8 nM

12 4 nM m m ~ a im a im mm

5 1 nM I I I I I I I

0 20 40 60

t ime, t x ( s ) Fig. 3. Time-course of Pi formation. ATP hydrolysis catalyzed by the active, reduced enzyme was measured at different initial ATP concentrations. The numbers at the slopes give the rates in pM s- i. For clearer presentation the data are shifted arbitrarily along the

ordinate.

292

gether with the site described by K~t = 220 /zM, at least three sites involved in ATP hydrolysis.

These data are obtained with different chloroplast preparations which differ in Vm~ x. When the relative

rates v/vm= are plotted the same functional dependence on ATP concentration is observed. The Pi formed from the [3'-32 P]ATP was measured after denaturation of the thylakoids. Therefore, the term Pi formed im-

.-., 0 .002

0%

"~ 0.001 2

¢-

O.

o 0 .000

A [CF.F, ]°= 20 nM

k,=1.5.10a M -I .=-* I

5 10

[ ATP] ( nM )

,.-., 0.010

to "~ 0.005 o & .

e-

13_

3

B [cF, F, ] ° - 2o .u

k"- 3.10= M-t ' l - 1 ~ ~ m

~ M - I .=-1 0.000 - ' '

0 5 10 15 20 25

[ ATP] ( nM )

~ 0 .020

to . ~ to o 0 .010

t- O.

"6 0.000 o - l ,- '

8

C [CFeFI] °,,- 20 nU

. / e I le'= 3" 1011d-l"u -1

i i !

20 40 60 80

[ ATP] ( ~u )

1.2 Ito

l :~ 0.8

?, 0.4 e.-

"6 o.o- 0.0

E

D [ CF, F, ]"= 20 nU

.1-1

i !

1.0 2.0 3.0

[ ATe ] (p,M)

I. 30

1o O.

~ o

E [CF. F,] °- 2O nU

i I i I i

-.-, 0 .0 0 .5 1.0 1.5 0 [ATP] ( mM )

Fig. 4. Rate of ATP hydrolysis as a function of the ATP concentration. Data from Fig. 3 and additional measurements. The open circles indicate data from Fig. 5. ATP concentration: (A) up to 10 nM; (B) up to 25 nM; (C) up to 90 nM. The solid line in (C) above 55 nM is the same function as in (D) extrapolated to lower concentrations. (D) ATP concentration up to 3.5/~M; (E) up to 1.5 mM. The solid line in (E) is calculated from

Michaelis-Menten kinetics (Vma x = 30 M A T P / ( M CFoF I s), K~ = 220 ~M).

15

• ~ O [ATP]°=SnM0= O ~ / , A ~ ' ~ O o [ ATP ] ~7 nU ¢..

O. 1G - o 1 0

o

• 5 k-

12-

0 10 20 30 40 50 60 70 80 90

t i m e , t x ( s )

Fig. 5. Time-course of the released free Pi. The free Pi was measured after removal of the enzyme-bound Pi by filtration ([CFoF]~'---20

nm). The numbers give the rate in n M s - t.

plies the concentration of enzyme-bound Pi and free Pi (see Fig. 3). If the enzyme is able to carry out many turnovers at high ATP concentration, then the enzyme-bound Pi can be neglected. However, when the substrate concentration is lower than the enzyme concentration, the concentration of enzyme-bound P~ may play a significant role. In an extreme case all of the Pi

remains bound to CFoF v This makes the interpretation of the kinetics as shown in Fig. 4 difficult. Therefore, the formed free Pi was measured after filtration of the thylakoids (see Materials and Methods). Fig. 5 shows the time-course of the formed free Pi for two ATP-concentrations indicating a linear increase with time. The slopes give the respective rates which are represented in Fig. 4A and C as open circles. Obviously, there is no significant difference between the rates based on the concentration of formed free Pi o r on the sum of the concentration of formed enzyme-bound and free P,. This implies that within the time resolution of these experiments (about 10 s), the steady-state concentration of enzyme-bound Pi is built up. This conclusion is

293

confirmed by direct measurements of the kinetics of formation of the enzyme ATP complex [38]. Therefore, the rates depicted in Fig. 4 indicate an increase of the formed free P~ in the whole concentration range from 1 nM to 1 mM ATP. It should also be mentioned that the free ATP concentration does not change signifi- cantly during the reaction time: within 40 s a decrease of approx. 30% of the initially concentration was found by measuring directly the decrease of free ATP at 4 nM [38] and at 30 nM [24].

Discussion

The inactivation of the active, reduced enzyme The kinetic analysis of ATP hydrolysis catalyzed by

the H +-ATPase from chloroplasts is difficult since the enzyme inactivates during the reaction if the experimental conditions are not properly adjusted. It is well known that upon activation ADP is released from the enzyme [21-23] and that rebinding of ADP under deenergized conditions inactivates the enzyme [26,31- 35]. With the washing procedure described in this work, the free and the bound ADP were nearly completely removed (see Table I). Nevertheless, the measurements shown in Fig. 2 indicate that the enzyme inactivates with a first-order rate constant ki, = 1.5" 10 -3 S - I , i.e., the half-lifetime of the active state is 450 s under our experimental conditions.

Obviously, E red inactivates by at least two different - - a

parallel reactions leading to different inactive, reduced states:

kl /

E ted

~ k2 , E[edADP

+ A D P

d[E=d] kt[Eraed]+k2[Eraedl[ADPl=kct.[E•ed ] dt

(1)

TABLE II

Rate constants for inactivation from literature

The rate constant k 2 was measured either by ADP rebinding or by decrease of the ATP hydrolysis activity, k I was measured by the decrease of ATP hydrolysis activity in the absence of ADP.

Half-lifetime Rate constants Reaction conditions Ref.

(~'(s)) kt ( s - l ) k2 (M_t s_l )

450 1.5-10- 3 990 7-10 -4

35 2.10- 2 70 1.10 -2

100 1.10 -2 1.105

100 7-10 -3 1.105

3.10 4 6.10 5 2.10 4

washed thylakoids (2 mM Pi, 2 mM Mg 2+ ) this work washed thylakoids (without Pi, without Mg 2+ ) 36 washed thylakoids (without Pi, 5 mM Mg 2+ ) 36 thylakoids (without Pi and Mg 2+ ) 36 thylakoids (2 mM Pi, 2 mM Mg 2+ ) 37 thylakoids (6 raM Mg 2+, without Pi) 31 isolated, reconstituted CFoF l 26

(kl: 5 mM Pi, 2 mM Mg2+; k2:2 mM Mg 2+ ) thylakoids (without Pi, 10 mM Mg 2+ ) 32 thylakoids (2 mM Mg 2+, without Pi) 33,34 thylakoids (without Pi, 5 mM Mg 2+ ) 35

294

with

k e n = k I + k 2 [ A D P ]

The inactivation by ADP-rebinding (second-order reaction) leads to an inactive enzyme with a tightly bound ADP, whereas the other inactive state contains no bound ADP. The concentrations of free ADP and free ATP during the inactivation were 3 nM and 0.1 nM respectively (see Table I). It can be calculated from the second-order rate equation with the rate constants for ADP binding (105 M - l s -1) and ATP binding (1.5 • 105 M-1 s - l ) that binding of both nucleotides is too slow to account for inactivation. Therefore, it is not possible that tightly bound ATP dissociates then rebinds to a catalytic site of the enzyme, hydrolyzes to ADP and P~ and inactivates the enzyme after P~ dissociation.

The rate constants for inactivation from recent literature data are collected together in Table II. These data show that the inactivation of the enzyme depends on experimental conditions, i.e., concentration of ADP, Pi, Mg 2÷, etc.

Appropr ia te experimental conditions require that the state of the enzyme is known and that it does not change during the experiment. This must always be checked for each set of experimental conditions. In this work the time range was limited to 60 s, where maximally 9% of the enzymes can inactivate in this time, and thus considered negligible in the further analysis.

Usually, an effective rate constant of inactivation or the corresponding half life time is measured (see Eqn. 1) and only a proper kinetic analysis reveals both reaction pathways.

Nucleotides bound to CFoF 1 The nucleotide content of CFoF j depends on the

preparat ion procedure. Isolated CF t contains up to six nucleotides [5,6]. CFoF 1 reconstituted by dialysis into asolectin liposomes contains one ATP and one ADP per CFoF ~ [26]. CFoF l bound at thylakoid membranes contains two ATP and one ADP per CFoF ~ (sec Table 1 and Ref. 24) Using the washing procedure described in this work, CFoF ~ contains after activation one bound ATP per CFoF ~ and the free nucleotide concentration is lowered to about 1 /10 of the enzyme concentration. This is necessary when reactions are to be measured at substrate concentrations lower than the enzyme concentrations (substoichiometric concentrations). With- out the removal of the bound ADP prior to the mea- surement one has always the problem that this nucleotide is released upon activation. Therefore, the free nucleotide concentration is at least equal to the enzyme concentration and it depends on the reaction conditions during and after the activation whether free ADP or free ATP is found [24].

Du and Boyer [36] have described a washing procedure for removing bound nucleotides. However, we

found 0.7 bound ADP per CFoF 1 when we used a similar procedure [38]. This difference indicates the importance of the membrane energisation during washing and centrifugation. This t reatment results in the complete release of the tightly bound ADP. The reaction conditions in (Ref. 36) are such that there is a nearly equivalent ratio of ADP to enzyme and the apparent rate constant for ADP rebinding is modified by Mg 2+ and Pi. We conclude that although the half- lifetime of inactivation is similar for both procedures (see Table II), the underlying mechanism is probably different. The inactivation described in our work occurs without ADP rebinding, since there is no ADP present.

The rate of A TP hydrolysis The rate of ATP hydrolysis can be described by a

Michaelis-Menten kinetics in the millimolar range with a K ~ = 220 ~ M and Vma x = 30 s - l (see solid line in Fig. 4E). This is in accordance with earlier results where K ~ values between 70 and 150/xM have been reported [15-19].

Below an ATP concentration of 7 nM, a linear increase of the rate with increasing substrate concentration is observed. Above 7 nM a supralinear increase is found and a plateau is reached between 25 and 55 nM. The supralinear increase requires the presence of at least two ATP binding sites. From the rate at the plateau ( I ;p l = 7 • 10 -3 s- l) and the data in Fig. 4C the substrate concentration for the half-maximal rate is obtained (15 nM) and we take this as the apparent K M value for these two sites. It is of course clear that the sigmoidal dependence of the rate up to 55 nM is not representative of Michaelis-Menten kinetics. However, for comparison with literature data wc characterize both sites by the substrate concentration at which the half-maximal rate is observed. In the simplest case, both ATP-binding sites are catalytic sites which arc intrinsically identical and occupation of both sites stim- ulate mutually their rate. However, binding of ATP to a non-catalytic site might increase the rate of ATP hydrolysis on the first catalytic site. In isolated CF~ the occupation of non-catalytic sites influences the rate of ATP hydrolysis [44-46]. This will also give rise to a supralinear increase in the rate with ATP concentration. On the basis of our results wc can not distinguish between the two possibilities.

Our data indicate, therefore, the participation of one catalytic site with K ~ = 15 nM, a regulatory site with the same dissociation constant and a catalytic site with K ~ = 220 /xM, or two cooperative catalytic sites with Kr~ = 15 nM and a third catalytic site with K~ = 220 /xM. The catalytic site with K~ = 220 # M is intrinsically not identical to the site(s) with K~ = 15 nM. We have shown earlier that the catalytic sitcs arc heterogeneous [24,43]. The site with K ~ = 220/xM has

a turnover of 30-90 s - t ; the site(s) with K~ = 15 nM has a turnover of 0.07-0.8 s- t depending on the occupation of other sites [24,43].

The observation of a constant rate at ATP concentrations between 25 nM and 55 nM (plateau) is un- usual. The most obvious explanation is that the rate at the plateau represents the maximal rate when two sites are cooperating. This possibility can be excluded. The complete occupation of two sites at an initial enzyme concentration of 20 nM would require 40 nM ATP. At the beginning of the plateau phase only 25 nM ATP is present initially, i.e., the enzyme to substrate ratio does not allow the complete occupation of two sites. Fur- thermore, it has been shown earlier that at 20 nM free ATP only a fraction, about 6%, of the enzyme is present as E-ATP complex and the turnover is about 0.8 s - l [24,43].

Another explanation would be the binding of ATP to a site which inhibits two-site ATP hydrolysis. It is well known that ATP (or ADP) binding to CFoF 1 with K o = 1 /zM inhibits the proton flux through the enzyme [39-41]. Since the K o is in the micromolar range it seems unlikely that this effect plays a role in the nanomolar range.

The third explanation is the existence of a slow conformational change which occurs after the binding of ATP. Proton transport coupled ATP hydrolysis of ATP to enzyme-bound or free products can take place only after the conformational change and this process might become rate-limiting. The conformational change might be connected with a protonation of the enzyme from the outside or with the transport of protons into the thylakoid lumen. Currently, this seems us the most plausible mechanism.

From the supralinear increase of the rate with increase ATP concentration we must conclude that above 7 nM ATP two cooperating sites are involved in ATP hydrolysis. Therefore, uni-site ATP hydrolysis can be observed only below 7 nM ATP at an enzyme concentration of 20 nM, i.e., the enzyme-to-substrate ratio must be higher than 3. The observation that the substrate concentration must be at least a factor of 3 lower than the enzyme concentration is in accordance with similar observations with MF 1 [13] and EF l [20]. CFoF 1 contains usually one tightly bound ADP per CFoF 1 which is released during activation. Therefore, the catalytic reaction after the activation is started with an equimolar ratio of enzyme to ATP (or ADP according to the reaction conditions [24]). In order to investigate uni-site ATP hydrolysis it is necessary to remove the tightly bound ADP, e.g., by the washing procedure described in this work.

Acknowledgments

This work has been supported by the Deutsche Forschungsgemeinschaft SFB 312 and by a scholarship

295

from the Studienstiftung des Deutschen Volkes to A.L. The authors thank Mrs. Sabine Pfafferott for typing the manuscript and Robert D. Kirch for critical read- ing.

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