Báo cáo Y học: The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F1-ATPase potx

The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F 1 -ATPase Noriyo Mitome, Sakurako Ono, Toshiharu Suzuki, Katsuya Shimabukuro, Eiro Muneyuki and Masasuke Yoshida Chemical Resources Laboratory, Tokyo Institute of Technology, Japan F 1 -ATPase i s inactivated by entrapment of MgADP in catalytic sites and reactivated by MgATP or P i . Here, using a mutant a 3 b 3 c complex of thermophilic F 1 -ATPase (aW463F/ bY341W) and monitoring nucleotide b inding by ¯uorescence q uenching of an introduced tryptophan, we found that P i interfered with the binding of MgATP to F 1 -ATPase, but binding of MgADP was interfered with to a lesser e xtent. Hydrolysis of MgATP by F 1 -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), a lso gave the same results. The half-maximal concentrations of P i that suppressed the MgADP-inhibited form a nd interfered with MgATP binding were both  20 m M .Itislikelythatthe presence of P i at a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme±MgADP±P i complex, an active intermediate in the catalytic cycle. Keywords: competition; F o F 1 -ATP synthase; MgADP inhibition; nucleotide binding; tryptophan ¯uorescence. F o F 1 -ATP synthase synthesizes ATP from ADP and inorganic phosphate (P i ) by using the energy of proton ¯ow driven by a transmembrane electrochemical proton gradient [1±3]. The enzyme c onsists of a c ytoplasmic domain, referred to as F 1 -ATPase or F 1 , which carries catalytic sites for the synthesis and hydrolysis of ATP, and a membrane integral domain, F o , which conducts protons across the membrane. F 1 has a subunit composition a 3 b 3 cde and can be reversibly separated from F o .Therearesix nucleotide-binding sites in F 1 -ATPase, thre e of which are catalytic sites, located on the b s ubunits, and three other noncatalytic sites, located on the a subunits. In the crystal structure of bovine mitochondrial F 1 -ATPase, the three a and three b subunits are arranged alternately like s egments of an orange around the central coiled-coil structure of the c subunit [4]. During ATP hydrolysis, F o F 1 -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 t he onset of this inhibition [11]. The crystal structure of mitochondrial F 1 -ATPase obtained in the presence of ADP, AMPPNP, and NaN 3 in 1994 [4] probably represents this MgADP- inhibited f orm. The MgADP-inhibited form i s a ctivated by ATP binding to the noncatalytic sites [12±15]. An apparent K d for the activation process was deduced to be 430 l M using nucleotide-depleted mito chondrial F 1 -ATPase [ 14]. Previously, we have reported that the mutant a 3 b 3 c subcomplex (DNC), which is defective in the noncatalytic nucleotide-binding site, is unable to continue ATP hydro- lysis b ecause all the subcomplexes remain in the M gADP- inhibited form [16]. However, the ATPase-inactive DNC F o F 1 -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 a 3 b 3 c subcomplex was activated by P i and showed continuous ATP hydrolysis activity in the presence of P i [18]. P i also activated the MgADP-inhibited form o f t he wild-type a 3 b 3 c subcomplex [18,19], F 1 [5,9] and F o F 1 -ATPase [11, 20, 21] in a similar manner. These studies suggested that P i bound to catalytic site(s) and suppressed formation of the MgADP-inhibited form. In this study, we tried to elucidate the mechanism by which P i prevents the formation of the MgADP-inhibited form and the relationship between P i binding and nucleotide binding at catalytic sites. For this purpose, we used the a (W463F)3 b (Y341W)3 c mutant subcomplex in which a Tyr r esidue near t he catalytic site (Y341) was replaced by Trp to monitor nucleotide binding to the catalytic sites. In addition, aW463 was replaced b y Phe to reduce t he 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 P i 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 P i ,inwhichP i at a catalytic site shifts the equilibrium from the MgADP-inhibited form to an enzyme±MgADP±P i 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 2 3 July 2001, revised 25 September 2001, ac cepted 22 October 2001 ) Eur. J. Biochem. 269, 53±60 (2002) Ó FEBS 2002 MATERIALS AND METHODS Strains, plasmids, and proteins Escherich ia c oli 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 c orresponding 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 t o pro- duce pKABG1-aW463F/bE190Q/bY341W. The s ubcomplexes 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 m M potassium phosphate buffer (pH 7.0) containing 1 m M EDTA. The puri®ed fraction was adsorbed on a Butyl-Toyopearl column (Tosoh), which was equilibrated with 100 m M sodium phosphate buffer (pH 7 .0) containing 50 m M Mops/KOH, 50 m M KCl, 1m M EDTA, 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 m M Mops/ KOH buffer (pH 7.0) containing 1 m M EDTA. The eluted enzyme was passed through a gel-®ltration column PD10 (Pharmacia Biotech) pre-equilibrated with 50 m M Mops/ KOH buffer (pH 7.0) containing 50 m M KCl and 2 m M MgCl 2 and used for the following experiments. The preparation contained less than 0.05 mol adenine nucleotide per mol enzyme. The ATPase a ctivity of t he enzyme preparation at 4 m M MgATPdependedontheKClconcentrationintheassay mixture. At 50 m M KCl, the activity was 12±13 U ámg )1 , whereas at 1 60 m M KCl, it was  30±40 Uámg )1 ,whichare in the normal range for thermophilic F 1 -ATPase activity. ATP hydrolysis assay ATP hydrolysis was measured using an ATP-regenerating system as a decrease in A 340 of NADH at 25 °C. The assay mixture contained 50 m M Mops/KOH buffer (pH 7.0), 50 m M KCl, 2.5 m M phosphoenolpyruvate, 4 m M ATP, 6m M MgCl 2 , 200 lgámL )1 pyruvate kinase, 200 lgámL )1 lactate dehydrogenase, and 0.2 m M NADH. 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 a 3 b 3 c 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 . T he ATPase activities at the initial phase (the slope of A 340 between3and13s)weremeasured. Fluorescence measurements Fluorescence was measured with a spectro¯uorometer (F4500; Hitachi) at 25 °C. After nucleotide depletion, a 3 b 3 c was diluted to 100 n M inacuvettetoatotalvolume of 1.2 mL in 50 m M Mops/KOH (pH 7.0) buffer containing 50 m M KCl and 2 m M MgCl 2 . The excitation wavelength was 295 nm and the emission wavelength was 345 n m. The slit for excitation was set at 5 nm and the slit for emission at 10 n m. T he 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 a fter the addition of MgATP s olutions. Calculation of total Mg 2+ concentrations At a high P i concentration, it is possible that free Mg 2+ and MgADP concentrations decrease because of complex formation between P i and Mg 2+ . It has previously been shown that the activating effects of EDTA and P i on the MgADP-inhibited enzyme are additive, indicating that activation by P i occurs via a m echanism other than simply reducing the Mg 2+ concentration [28]. However, in order to exclude the possibility o f reducing MgADP at h igh concentrations of P i , we adjusted the total Mg 2+ concen- tration for each P i concentration in the relevant experi- ments. The c oncentration of e ach ionic s pecies was calculated as described by Clark et al. [29]. RESULTS ATPase activity of the a (W463F)3 b (Y341W)3 c subcomplex after incubation with MgADP The a (W463F)3 b (Y341W)3 c subcomplex was preincubated w ith MgADP at various molar r atios 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)3 b (Y341W)3 c subcomplex reached 1.5 : 1. It has previously been reported that the DNC a 3 b 3 c subcomplex was inhibited b y MgADP at a 1 : 1 molar ratio [16]. In a similar expe riment, wild-type a 3 b 3 c complex w as inhibited by MgADP at a ratio of 1 : 1.5 (E. Muneyuki, unpublished results). Here we conclude that the a (W463F)3 b (Y341W)3 c subcomplex assumes the MgADP-inhibited form in the same manner as the wild-type a 3 b 3 c complex. Effect of P i on the MgADP-inhibited form We have previously reported th at the DNC a 3 b 3 c sub com - plex did not exhibit steady-state ATP hydrolysis in the absence o f P i [16], but signi®cant steady-state ATP hydro- lysis was later observed i n the presence of P i [18]. Here we examined the effects of P i on the formation of the MgADP- inhibited f orm o f the a (W463F)3 b (Y341W)3 c subcomplex. The extent of ADP inhibition of the a (W463F)3 b (Y341W)3 c sub- complex after 5 m in preincubation with stoichiometric concentrations of MgADP plus various concentrations of potassium phosphate was measured. Increasing concentra- tions of P i prevented the formation of the MgADP-inhibited form of the a (W463F)3 b (Y341W)3 c subcomplex (Fig. 2A). The P i concentration that yielded half-maximal prevention of inhibition was 21  6.4 m M (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 m M ) in the preincubation medium had no protective effect against MgADP inhibition. When the P i concentra- 54 N. Mitome et al.(Eur. J. Biochem. 269) Ó FEBS 2002 tionwaskeptat20m M and the ratio of MgADP to the subunit complex in the preincubation mixture was changed, the e xtent o f i nhib ition by MgADP a lso d ecreased (Fig. 1 B). From these results, we conclude that P i prevented MgADP inhibition under these conditions. This is consis- tent with the previous report that inactivation of F 1 -ATPase as a result of the MgADP inhibition could be partially prevented by P i [18]. The above results indicate that the a (W463F)3 b (Y341W)3 c subcomplex has the same properties for MgADP inhibition and P i activation as t he wild-type complex, allowing us to investigate the effect of P i on MgADP inhibition. Effect of P i on MgADP binding to catalytic sites Formation o f t he MgADP-inhibited form is caused by entrapment of inhibitory MgADP in a catalytic s ite [5±9]. After prior loading of a catalytic site of MF 1 [14,30], TF 1 [31], and chloroplast F 1 -ATPase [32] with MgADP, the enzymes hydrolyse ATP with an extended lag phase. The protective effect of P i against ADP inhibition described above may be explained by interference of MgADP binding by P i . 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 i n the presence of P i 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 p rior incubation with MgADP. (A)TimecourseofATP 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 so lution s was withdrawn a nd 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 concentratio n constant, MgCl 2 concentrations in the preincubation mixturewereadjustedtobe2m 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 p lotted 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 . Preven tion of th e formation of th e MgADP-inhib ited form by phosphate. (A) Tim e c ourse 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 o f variou s con centrations o f p otassium ph osphate. Th e 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+ concentration s added as M gCl 2 in the preincubation mixture were adjusted t o 3.0, 3.5, 4.5, 6.5, 10, and 16 m M for0,2.5,10,25,50and100m M potassium phosphate to keep the MgADP concentration constant. Afte r th e pre incubation, 40 lLof 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) De pendence of t he relative ATP hydrolysis activity of the a (W463F)3 b (Y341W)3 c su bcomplex o n pr eloaded potassium phosphate concentration. The residual A TPase activities during the initial phase (the s lo pe of A 340 between3and13s)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 ab sence of P i ,andb 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 . Ó FEBS 2002 Suppression of MgADP inhibition by phosphate (Eur. J. Biochem. 269)55 residues. On addition of saturating amounts of nucleotide (1 m M MgADP or 1 m M MgATP), the ¯uorescence of the three introduced tryptophans was completely quenched. Figure 3A shows t he time course of the ¯uorescence change on addition of MgADP. In the experiments, the concentrations of the a (W463F)3 b (Y341W)3 c subcomplex and MgADP were 100 n M and 300 n M , respectively. Contrary to our expectation, the presence of P i had little effect o n the binding of ADP to the catalytic sites. Quenching was essentially complete within 20 s. Actually, t he rate of the MgADP binding was slightly slower at higher P i concen- trations; however, t he magnitude of ¯uorescence quenching was a ffected little (Fig. 3C) i n t he P i concentration range in which the protective effect o f P i was most prominent. T hese results sugges t that P i prevents MgADP inhibition, although MgADP remains bound to the catalytic sites. Effect of P i on MgATP binding to catalytic sites Previously, we suggested that P i acts at a catalytic site to prevent ADP inhibition [18]. However, given that, P i did not interfere with ADP binding at any of the catalytic sites, there are two possible explanations of our r esults. One i s that P i 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 P i binds to a site o ther than any of the catalytic sites. In the ®rst case, it is possible that P i binding interferes in ATP binding, because of the c phosphate. To con®rm this possibility, MgATP binding to the catalytic sites was monitored b y ¯uorescence quenching of the introduced tryptophan residues in the presence of P i . The time course of the ¯uorescence quenching on addition of MgATP in the presence of various concentrations of P i is shown in Fig. 3B. Although the ®nal magnitude of the ¯uorescence quenching by the addition of MgATP d id not signi®cantly depend on the P i concentration (Fig. 3C), ATP binding to t he cata lytic sites was generally slower at higher P i concentrations. However, the result was somewhat ambiguous, possibly because of the hydrolysis of ATP to A DP during the experiment. To clarify this point, the a (W463F)3 b (E190Q/ Y341W)3 c subcomplex, which was able to bind MgATP b ut unable to h ydrolyse MgATP [ 27], was u sed. In the case of the E. coli F 1 -ATPase, a mutant equivalent to a 3 b (E190Q/ Y341W)3 c displayed a nucleotide-binding pattern that was similartothatofthea 3 b (Y341W)3 c subcomplex [24]. Figure 4B demonstrates the time course of ATP binding to the a (W463F)3 b (E190Q/Y341W)3 c complex in the presence of P i . Compared w ith Fig. 3B, Fig. 4B indicates more clearly that ATP bound to the catalytic sites more s lowly a t higher P i concentrations. Two time constants were estimated by ®tting a double-exponential function to the data and plotted against P i concentration (Fig. 4C). The time resolution of our experimental system was, however, not good enough for the fast phase and we d id not attempt to analyze it. On the other h and, the slow t ime constant of MgATP binding was clearly longer at higher P i concentrations. The apparent af®nity of P i was estimated to be 19  3.6 m M by ®tting a simple Micha elis±Menten equation to the data for the slow time constant. This value was similar to th e K d of 21 m M measured for suppression of MgADP inhibition (Fig. 2B). On the other hand, the presence of P i had little in¯uence on the binding of ADP to the catalytic sites (Fig. 4A,D). Again, P i had little effect on the time constant or magnitude of ¯uorescence quenching by MgADP binding in the P i concentration range where the effect of P i was most prominent for the a (W463F)3 b (Y341W)3 c complex. From these results, we conclude that P i is bound at a catalytic site of the a (W463F)3 b (Y341W)3 c subcomplex where it competes with Fig.3.EectofP 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,andthe 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 experimen tal procedure is the same as i n (A), except that MgATP was added instead of MgADP. (C) The amplitude of ¯uorescence quenching upon addition of MgATP (d)andMgADP (j) p lotted against P i concentration. The am plitudes were rea d from (A) and (B). 56 N. Mitome et al.(Eur. J. Biochem. 269) Ó FEBS 2002 MgATP. Close coincidence of the apparent af®nity of P i that interferes with ATP b inding (19 m M ) and the apparent af®nity that prevents formation of the MgADP-inhibited form (21 m M ) suggests that the P i that prevents MgADP inhibition is located in a catalytic site even though it does not signi®cantly affect ADP b inding to that catalytic site. Titration of MgATP binding to the a (W463F)3 b (E190Q/Y341W)3 c complex Figure 5A shows MgATP binding to the a (W463F)3 b (E190Q/ Y341W)3 c complex in the presen ce or absence of 10 m M phosphate. As shown in Fig. 5A,B, MgATP binding was fast in the absence of phosphate, b ut slow in the presence of 10 m M phosphate at appropriate MgATP concentrations. At 200 n M MgATP, the binding was slowest. When MgATPwaslowerorhigherthan200n M ,theMgATP binding was f ast (Fig. 5A,C). At 1 l M , the ra te of MgATP binding in the presence of P i was faster than at 300 n M .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 n M , t he 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 P i under bi-site conditions where MgATP binds to the second catalytic site of the enzyme. The above results suggest that P i inhibits ATPase activity, particularly at low ATP concentrations. We t hen compared the initial ATPase activity of the a (W463F)3 b (Y341W)3 c com- plex in the presence a nd absence of 20 m M P i . Although there was not a p recise agreement with the d ata i n Fig. 5C, which was obtained using the a (W463F)3 b (E190Q/Y341W)3 c complex, the ATPase activity was indeed 29, 48, and 13% inhibited at 0.3, 1, a nd 10 l M ATP in the presence of P i .At 100 l M ATP, there was no apparent inhibition by P i . DISCUSSION P i suppresses formation of the MgADP-inhibited form. There may be several explanations. The simplest is that P i binds to a catalytic site, and inhibitory ADP is ejected from it. The second is that P i and ADP bind simultaneously to the catalytic site and the enzyme is kept active. The third possibility is t hat P i binds to noncatalytic sites (or any other site) w hile ADP binds to the catalytic site. The fourth possibility is that P i binds to the noncatalytic sites (or any other site) and inhibitory ADP i s ejected from the c atalytic sites. The last possibility is reminiscent of the situation in which ATP binding to the noncatalytic sites activates F 1 from the MgADP-inhibited form [12±15]. Two important points are whether ADP remains bound to the catalytic sites when P i is added to activate the ADP-inhibited enzyme, and the location of P i binding. When P i was added to the assay mixture, however, the effect of P i in relieving MgADP inhibition was not clear. This may be because P i in the assay mixture simultaneously acts as competitive inhibitor of ATP hydrolysis and suppresser of MgADP inhibition. In the case of EF 1 ,P i was shown to inhibit uni- site and bi-site ATP hydrolysis [33]. Therefore, we mainly examined the effect of P i on MgADP inhibition by adding P i to the preincubation mixture with ADP and observing the resultant ATPase activity and ¯uorescence quenching. From our results, we conclude that the presence of P i at 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 p resence of P i . The reaction m ixt ure 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 o f t he time constant of the MgATP binding (s)tothea (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 d rawn by ®tting the e quation a[P i ]/(K d +[P i ]) to the data points. Here, a is the maximum time constant in the presence of P i ,andK 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)andMgADP(j)plottedagainstP i concentration. Ó FEBS 2002 Suppression of MgADP inhibition by phosphate (Eur. J. Biochem. 269)57 inhibited form even while MgADP is still bound at that catalytic s ite. It is known that a number of anions exert various complex e ffects on F 1 -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 P i prevents MgADP i nhibition, it is highly likely that the presence of P i at a catalytic site suppresses the formation of the MgADP-inhibited state. The recently reported crystal structure of mitochondrial F 1 - 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 A TP at a catalytic site [38]. A s previously proposed [18], t he presence of P i at a catalytic site m ay shift t he equilibrium from the MgADP- inhibited form to the enzyme±MgADP±P i complex, which is an active intermediate in the catalytic cycle. Maximum protection from ADP inhibition by P i was only 69% (Fig. 2 B), suggesting that there may be other inactive enzyme±MgADP±P i complexes, and, even in the presence of excess P i , 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 P i and membrane energization may fully protect the enzyme from ADP inhibition under phosphorylating co n- ditions. The apparent K d of P i that interferes with ATP binding (19 m M ), or that which prevents formation of the MgADP-inhibited form ( 21 m M ), was somewhat higher than the apparent K m for ATP synthesis by thermophilic F o F 1 -ATPase reconstituted into proteoliposomes (6±9 m M ) [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 P i . 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 P i 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 l M a (W463F)3 b (Y341W)3 c and 0.3 l M MgATP, the ®nal ¯uorescence level induced by MgATP binding was not signi®cantly in¯uenced by the presence or absence of P i (Fig.3B,C).OnlyaP i -dependent change in the rate o f MgATP binding was detected (Fig. 3B). Unfortunately, the result was somewhat unclear, probably because of hydro- lysis of MgATP during the exp eriment, therefore we used the a (W463F)3 b (E190Q/Y341W)3 c complex, which is unable to hydrolyse ATP. The ®nal ¯uorescence level quenched by MgATP binding in this case was also almost independent of P i (Fig. 4 B,D). T he retardation o f ATP binding in the presence of P i was clearly exhibited (Figs 4B,C ) and it was found that this retardation was observed over a limited ATP concentration range (0.1 l M <[ATP]<1l M , Fig. 5A,C). The inhibition of ATPase activity at low ATP concentration by 20 m M P i is qualitatively consistent with the retarded ATP binding. Below 0.1 l M ATP, the low signal to noise ratio i n 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 o f P i . I n t he concentration r ange in which retardation of ATP binding by P i was observed, ATP binding to the second catalytic site (bi-site) occurred. It is tempting to conclude that P i competes with ATP at the second catalytic site. It may i mply t hat the ®rst site favors ATP binding whereas the second site favors P i 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 P i on the ATP- binding rate was not observed. At high ATP concentrations, however, ATP binding is inherently fast, and the apparent absence of th e effect of P i on 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 o f t he catal ytic s ites 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 addedat0s.(B)TimecourseofMgATPbindingintheabsenceofP 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 M gATP concentrations in t he presence of P i .Thetime constants were estimated by ® tting a double- exponen tial function to thetimecourseofMgATPbinding.(d)Fasttimeconstants;(j)slow time constants. 58 N. Mitome et al.(Eur. J. Biochem. 269) Ó FEBS 2002 ACKNOWLEDGEMENTS 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. 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Biochem. 269) Ó FEBS 2002 . The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F 1 -ATPase Noriyo Mitome, Sakurako Ono, Toshiharu. 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