©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Location and Properties of Pyrophosphate-binding Sites in Escherichia coli F-ATPase (*)

Joachim Weber , Alan E. Senior

From the (1) Department of Biochemistry, University of Rochester Medical Center, Rochester, New York 14642

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Binding of pyrophosphate (PP) to the three catalytic (``C'') and three noncatalytic (``NC'') nucleotide sites of Escherichia coli F-ATPase was determined by fluorescence spectroscopy using mutant enzymes with tryptophan inserted specifically in either C sites (Y331W) or NC sites (R365W). Fluorescence of the tryptophan is quenched on binding of nucleotide; PP binding parameters were determined by competition with ATP or adenyl-5`-yl imidodiphosphate. It was found that MgPP binds to each NC site with K = 20 µM. In contrast, even at millimolar concentration, neither MgPP nor free PP showed significant binding to C sites. We confirmed that free PP displaces nucleotide from C sites, but this was shown to be due to complexation of Mg ions rather than to occupancy of the sites. MgPP bound at NC sites was found not to affect ATP hydrolysis rates. From the data we propose a two-phase model for nucleotide binding at NC sites. In phase one, NC sites recognize the pyrophosphate ``end'' of the nucleotide, which binds initially with K similar to MgPP; in phase two, a slow conformational change occurs which tightly sequesters adenine nucleotide. Phase two does not occur with guanine nucleotide. This model explains the preference of NC sites for adenine nucleotides.

P (5 mM) did not bind to either C or NC sites.


INTRODUCTION

Escherichia coli FF-ATP synthase is the enzyme responsible for ATP synthesis in oxidative phosphorylation and generation of a transmembrane proton gradient by ATP hydrolysis. It consists of a membrane-embedded F sector attached by a stalk to the F sector, which projects from the membrane on the cytoplasmic face of the bacterial plasma membrane (for recent reviews, see Senior, 1990; Fillingame, 1990; Capaldi et al., 1994). The F sector may readily be released from membranes in low ionic strength buffer and purified to homogeneity as an active ATPase (e.g. Senior et al., 1979).

Purified E. coli F-ATPase contains a total of six nucleotide-binding sites, of which three are catalytic, and three are relatively non-exchangeable and noncatalytic (Wise et al., 1983; Perlin et al., 1984; Issartel et al., 1986). As reviewed in Weber et al. (1993a), earlier affinity and photoaffinity labeling experiments and mutagenesis studies in several laboratories had suggested that residue Tyr-331() of the F -subunit was located in the catalytic site, in close proximity to bound ATP. Use of the fluorescent substrate analogs lin-benzo-ADP and lin-benzo-ATP in our laboratory indicated that residue -Tyr-331 makes direct contact with the base moiety of bound analog (Weber et al., 1992). Substitution of tryptophan, in the Y331W mutant, generated an F enzyme which retained catalytic activity similar to that of wild-type, and the fluorescence of the 3 -Trp-331 residues (in the three -subunits) provided a specific and direct probe of nucleotide binding to catalytic sites (Weber et al., 1993a). By this technique it was shown that all three catalytic sites must be occupied to achieve physiological (V) rates of ATP hydrolysis and that occupation of two sites leads, at most, to a very slow rate of ATP hydrolysis. Also, binding parameters for MgATP, MgADP, and MgAMP-PNP() at each of the three catalytic sites were reported; with all three nucleotides, the catalytic sites showed negative binding cooperativity. In further work (Weber et al., 1994a) the Mg dependence and nucleotide specificity of the catalytic sites and effects of inhibitors and mutations were established. X-ray crystallography studies of the bovine mitochondrial F-ATPase enzyme have recently defined the structure and environment of the three catalytic sites in detail and have confirmed that residue -Tyr-331 (actually -Tyr-345 in bovine) does indeed lie immediately adjacent to the purine ring of bound nucleotide (Abrahams et al., 1994).

A parallel line of reasoning and experimentation allowed us to characterize the noncatalytic sites. Residue Arg-365 of the -subunit appeared from sequence comparison to be homologous to residue -Tyr-331, and by mutagenesis and photoaffinity-labeling studies we confirmed the location of residue -Arg-365 as part of the noncatalytic site (Weber et al., 1993b). F obtained from the R365W mutant was found to retain catalytic activity similar to wild-type, and in this enzyme the newly inserted tryptophan provided a direct and specific fluorescent probe of nucleotide binding at noncatalytic sites (Weber et al., 1994b). Using this signal we established the Mg dependence, nucleotide specificity, and nucleotide-binding parameters at noncatalytic sites and demonstrated that occupancy of noncatalytic sites is not required for ATP hydrolysis. X-ray crystallography studies of the bovine mitochondrial F-ATPase enzyme have since defined the structure and environment of the noncatalytic sites in detail and have confirmed that residue -Arg-365 (actually -Arg-362 in bovine) lies immediately adjacent to the adenine ring of bound nucleotide (Abrahams et al., 1994).

Pyrophosphate (PP) is potentially a valuable probe of functional characteristics of F-ATPase nucleotide-binding sites in wild-type and mutant enzymes. Studies with bovine mitochondrial enzyme have demonstrated that up to 3 mol of [P]PP may become bound per mol F (Kironde and Cross, 1986; Issartel et al., 1987; Peinnequin et al., 1992; Jault and Allison, 1993). In the paper of Kironde and Cross(1986), PP was shown to promote release of adenine nucleotide from catalytic sites, and it was concluded that PP bound to two catalytic sites ( of Kironde and Cross(1986)). However, later work from the same laboratory (Milgrom and Cross, 1993) has indicated that PP became bound to noncatalytic sites and appeared not to compete effectively with GTP, ATP, or ADP for binding to catalytic sites. Jault and Allison(1993) also made the interpretation that PP binds at noncatalytic sites, whereas Peinnequin et al. (1992) concluded that PP binds to specific sites which interact with and influence occupancy of catalytic and noncatalytic sites, but which are distinct from both. Therefore, the current literature is conflicting as to the location of PP-binding sites. In bovine mitochondrial, F binding constants (K ) for PP of approximately 1-20 µM (Issartel et al., 1987; Peinnequin et al., 1992), or 15-230 µM (Kironde and Cross, 1986) have been reported. Studies of interaction of PP with E. coli F have been fewer. A recent paper (Hyndman et al., 1994) showed that PP competed effectively for binding of GTP or ATP to noncatalytic sites, but had little or no effect on binding to catalytic sites. PP binding parameters have not been reported for E. coli F.

The availability of mutant E. coli enzymes with reporter tryptophans substituted specifically at the catalytic or the noncatalytic sites provides a new technique which can be used to study PP binding. In this work, we have used the technique to study the sites at which PP competes with nucleotide for binding and to determine K values for PP. The data demonstrate a major difference between catalytic and noncatalytic sites and suggest that, in the binding process, the noncatalytic sites recognize initially the pyrophosphate ``end'' of nucleotide ligands. We offer also an explanation for the effect of PP to promote net loss of nucleotide from catalytic sites, based on its ability to complex Mg ions rather than by direct competition for occupancy of the sites.


MATERIALS AND METHODS

Fluorescence Measurements

These were done as described in Weber et al. (1993a, 1994a, 1994b). The buffers used in fluorescence experiments are described in the figure legends and in the text. Before use F enzymes were pre-equilibrated in buffer in one of two ways as follows. 1) Mg-free buffer: the enzyme was passed sequentially through two 1-ml Sephadex G-50 centrifuge columns in 50 mM Tris-SO, pH 8.0, at 23 °C. 2) Mg-containing buffer: the enzyme was passed through one 1-ml Sephadex G-50 column in 50 mM Tris-SO, pH 8.0, 2.5 mM MgSO, at 23 °C.

F Purification and Characterization: Assays of Enzyme Function

These were done as described in Weber et al. (1994b). The procedure for preparation of nucleotide-depleted enzyme was as in Senior et al.(1992).

F Enzymes

Enzyme from strain SWM4 (Y331W mutant) was as in Weber et al. (1993a); from strain AW7 (R365W mutant) as in Weber et al. (1994b); from strain SWM1 (wild-type) as in Rao et al. (1988a). Strain CB5 was constructed as follows. Plasmid pOW1 (Wilke-Mounts et al., 1994) was digested with SstI and the larger (vector) fragment was purified. Plasmid pSWM4 (Weber et al., 1993a) was digested with SstI, and the smaller fragment containing the Y331W mutation was purified. The two purified fragments were ligated together and transformed into strain TG1rA (Rao et al., 1988b). Plasmid minipreps obtained from transformants were screened first with PstI to determine correct orientation of the SstI-SstI insert, and then with NaeI, which identifies the Y331W mutation. One plasmid, named pCB5, was subjected to DNA sequencing to confirm that the Y331W mutation was present, and then transformed into strain AN888 (Downie et al., 1981), yielding strain CB5. Plasmid pCB5 contains six total mutations. In addition to the Y331W mutation at the catalytic site, it contains the mutations W28L/W513F/W108Y/W206Y/W107F which together remove all the tryptophans naturally present in wild-type F.


RESULTS

F Enzymes Used in This Work

SWM4 F (Y331W mutant) contains the 9 wild-type tryptophans plus 3 additional tryptophans at residue -331. The properties of this enzyme are similar to wild-type and have been described by Weber et al. (1993a, 1994a). The -Trp-331 fluorescence signal reports occupancy of the catalytic sites by nucleotide. AW7 F (R365W mutant) contains the 9 wild-type tryptophans plus 3 additional tryptophans at residue -365. The properties of this enzyme are also similar to wild-type and have been described by Weber et al. (1994b). The -Trp-365 fluorescence signal reports occupancy of the noncatalytic sites by nucleotide.

CB5 F contains none of the wild-type tryptophans, it contains only 3 tryptophans at position -331 (see ``Materials and Methods'' for construction of strain CB5). The entire fluorescence signal ( = 295 nm) in this enzyme is due to -Trp-331 at the catalytic sites. Strain CB5 showed the same growth characteristics as wild-type on succinate plates and in liquid medium containing limiting (3 mM) glucose. CB5 F was found to show normal molecular size by Sephacryl S-300 chromatography and normal subunit composition on SDS-gels. V for ATP hydrolysis was 70% of wild-type value, and k/K (1.8 10M s) was similar to that of wild-type (1.9 10M s).

Studies of Interactions of PP with Catalytic Sites

Binding of Free ATP to Catalytic Sites in Presence and Absence of 1 mM Free PP

In these experiments, SWM4 F (Y331W) was used. The enzyme was pre-equilibrated with Mg-free buffer by passage through two centrifuge columns (see ``Materials and Methods''). The residual adenine nucleotide occupancy at catalytic sites was 0.2 mol/mol, as indicated by the fluorescence signal. Fluorescence spectra were recorded in EDTA-containing buffer, and different concentrations of ATP were added to elicit varying degrees of catalytic site occupancy, as indicated by quenching of the fluorescence signal. The fluorescence responses to ATP were similar to those reported previously (Weber et al., 1994a), and it may be noted that it was also shown previously that ATP hydrolysis activity was essentially zero under these conditions. The presence of 1 mM NaPP had no effect on the ATP-induced fluorescence responses (Fig. 1) indicating that free PP did not bind to catalytic sites.


Figure 1: Effect of free PP on binding of free ATP to the catalytic sites of E. coli F. SWM4 F (Y331W) was titrated with ATP in a buffer containing 50 mM Tris-SO, 0.5 mM EDTA, pH 8.0, either in absence () or in presence () of 1 mM NaPP. ATP binding stoichiometries were calculated from the decrease in fluorescence of residue -Trp-331. The solid line represents a fit to the data points obtained in absence of NaPP (2.9 equivalent binding sites with K = 71 µM).



Binding of MgAMP-PNP to Catalytic Sites in Presence and Absence of 1 mM MgPP

In these experiments, SWM4 F (Y331W) was titrated with AMP-PNP in a buffer containing 2.5 mM Mg. The enzyme was pre-equilibrated with Mg-free buffer by passage through two centrifuge columns, such that the residual adenine nucleotide occupancy at catalytic sites was 0.2 mol/mol, as indicated by the fluorescence signal. The binding curve for MgAMP-PNP seen in absence of MgPP (Fig. 2) was the same as that reported previously in Weber et al. (1993a). The presence of 1 mM MgPP had very little, if any, effect on the MgAMP-PNP binding curve (Fig. 2). summarizes the binding parameters obtained from the Fig. 2data. There was no significant MgPP binding to the two catalytic sites of lower affinity. In the case of the high-affinity site, we cannot exclude the possibility of very weak MgPP binding; however, the dissociation constant of this site for MgPP is at least 1 mM. Attempts to obtain more precise data by increasing the MgPP concentration failed due to formation of precipitate in the cuvette.


Figure 2: Effect of MgPP on binding of MgAMP-PNP to the catalytic sites. SWM4 F (Y331W) was titrated with AMP-PNP in a buffer containing 50 mM Tris-SO, 2.5 mM MgSO, pH 8.0, either in absence () or in presence () of 1 mM MgPP (1 mM NaPP plus 1 mM MgSO, increasing the total MgSO concentration to 3.5 mM). MgAMP-PNP binding stoichiometries were calculated from the decrease in fluorescence of residue -Trp-331. The solid line represents a fit to the data points obtained in absence of PP, the dashed line to those obtained in its presence. The resulting binding parameters are given in Table I.



Apparent Displacement of Catalytic Site-bound Nucleotide by PP

When SWM4 F was pre-equilibrated by passage through a single centrifuge column in buffer containing Mg ions (2.5 mM) the residual adenine nucleotide occupancy at catalytic sites was 0.4-0.8 mol/mol F, as indicated by the fluorescence signal. We previously reported (Weber et al., 1993a) that addition of 3 mM NaPP to this enzyme, in a buffer containing 2.5 mM Mg ions, caused an increase in fluorescence (by 5-10%), indicating that PP caused displacement of adenine nucleotide from catalytic sites. This confirmed the work of Kironde and Cross(1986), who showed that, in bovine mitochondrial F, 5 mM NaPP displaced adenine nucleotides from catalytic sites when added to enzyme in a buffer containing 2 mM Mg ions. Given that neither free PP nor MgPP competes for binding of nucleotide to catalytic sites (above), it appeared that the displacement of nucleotides from catalytic sites was not due to actual occupancy of catalytic sites by PP.

We carried out further experiments to investigate this phenomenon. CB5 F was used in this part of the work because in this enzyme the entire fluorescence signal ( = 295 nm) is attributable to the substituted -Trp-331; all of the wild-type tryptophans have been removed by mutagenesis. This eliminates any possibility that fluorescence changes engendered by PP are due to effects on tryptophans other than at residue -331. CB5 F was first pre-equilibrated by passage through two centrifuge columns in Mg-free buffer, after which it was found to contain 0.2 mol/mol adenine nucleotide at catalytic sites as indicated by the fluorescence signal. Fig. 3shows the fluorescence spectrum of this enzyme in absence of added nucleotide (curve 1). It may be remarked that this spectrum is very similar to the difference spectrum seen when comparing SWM4 F to wild-type enzyme (as in Fig. 2B of Weber et al. (1993a)), confirming that the fluorescence spectrum of SWM4 F is indeed the sum of the independent contributions of the residue -331 tryptophans and those present in wild-type F; this means that no significant fluorescence energy transfer occurs between both sets of the fluorophore. Addition of 1 mM MgATP to CB5 F (Fig. 3, curve 2) caused almost complete quenching of the fluorescence signal.() Addition of 1 or 10 µM MgATP caused partial quenching of fluorescence (curves 3 and 5) indicating catalytic sites occupancy of 1.2 and 1.9 mol/mol, respectively. The presence of 1 mM MgPP did not change the response to 1 µM MgATP (curve 4) or 10 µM MgATP (curve 6), demonstrating that MgPP did not compete with MgATP for binding at catalytic sites.


Figure 3: Effect of MgPP on ATP-induced quenching of CB5 F fluorescence. Shown are uncorrected emission spectra ( = 295 nm) of CB5 F (50 nM in 50 mM Tris-SO, 2.5 mM MgSO, pH 8.0). Curve 1 without added nucleotide (±1 mM MgPP); curve 2 with 1 mM ATP; curves 3 and 4 with 1 µM ATP; curve 5 and 6 with 10 µM ATP. 1 mM MgPP was present in curves 4 and 6.



CB5 F was then prepared by pre-equilibration in buffer containing 2.5 mM Mg ions by passage through one centrifuge column. This preparation retained about 0.8 mol/mol adenine nucleotide at catalytic sites as judged by the fluorescence signal. Addition of NaPP (3 mM final concentration) to this enzyme in buffer containing 2.5 mM Mg ions increased the fluorescence signal by 11-15%, indicating that release of nucleotide from catalytic sites had occurred. A 20-23% increase in fluorescence was obtained by addition of EDTA, instead of NaPP, to a final concentration of 3 mM and addition of EDTA to a final concentration of 6 mM increased the fluorescence by a total of 30%. The final level of the fluorescence signal in the latter case indicated complete emptying of catalytic sites.

In contrast, when NaPP and MgSO (both 3 mM) were added together, the fluorescence increase seen was smaller (6-8%) than with NaPP alone. The same final level of fluorescence was reached if the MgSO was added after the NaPP. Attempts to achieve a higher excess of Mg concentration over PP were confounded by precipitation in the cuvette, which caused an artifactual increase in the fluorescence signal. When EDTA and MgS0 (both 3 mM) were added together, the fluorescence increase seen was only 6% as contrasted to 20-23% with EDTA alone, and if 10 mM MgS0 was added with 3 mM EDTA, there was no increase in fluorescence at all.

These experiments indicated that displacement of nucleotides from F catalytic sites by PP is related not to actual binding of PP at the sites but rather to its ability to complex Mg ions. This is reasonable, given that the nucleotide species bound at catalytic sites in native enzyme after purification is ADP (Kironde and Cross, 1986; Senior et al., 1992), that the stability constants at pH 8.0 for MgADP and MgPP complexes are similar (pK(MgADP) = 3.6, pK(MgPP) = 3.7; see O'Sullivan and Smithers, 1979; Dawson et al., 1984), and that the affinity of catalytic sites for adenine nucleotide is strongly enhanced in presence of Mg (Weber et al., 1994a). Since the effect of PP may be readily mimicked by EDTA, and the enzyme prepared under the conditions described above contains noncatalytic sites essentially fully occupied by endogenous nucleotide (Weber et al., 1994b), this effect of PP does not involve the noncatalytic sites.

Studies of Interactions of PP with Noncatalytic Sites

Binding of MgAMP-PNP to Noncatalytic Sites in Presence and Absence of PP or P

Since binding of nucleotides to noncatalytic sites is Mg-dependent (Weber et al., 1994b), we did not conduct any studies in absence of Mg ions. Nucleotide-depleted AW7 enzyme (R365W mutant) was prepared, pre-equilibrated with buffer containing 2.5 mM Mg, and titrated with AMP-PNP either in the absence of, or in the presence of, 5 mM P, 100 µM PP or 200 µM PP. Fig. 4 shows the data obtained. The data were fitted according to a binding model assuming N equivalent and independent binding sites for MgAMP-PNP. A good fit was obtained, in absence of P or PP, with N = 2.8 and K = 16 µM. Previously, we found that the same binding model (3.0 and 2.8 equivalent sites, respectively) described well the binding of MgATP (K = 25 µM) and MgADP (K = 24 µM) to noncatalytic sites of AW7 F (Weber et al., 1994b). It is seen that 5 mM P had zero effect on MgAMP-PNP binding (Fig. 4, open squares), whereas 100 µM PP (closed circles) and 200 µM PP (triangles) competed for binding at all three sites. The apparent K (MgPP) calculated from the data in Fig. 4was 20 µM at each site (average of all data). Thus, it was evident that the K (MgPP) was similar to that for Mg nucleotides.


Figure 4: Effect of P and MgPP on MgAMP-PNP binding to the noncatalytic sites. Nucleotide-depleted AW7 F (R365W) was titrated with AMP-PNP in a buffer containing 50 mM Tris-SO, 2.5 mM MgSO, pH 8.0; () in absence of P or PP; () with 5 mM P, () with 100 µM PP, () with 200 µM PP. MgAMP-PNP binding stoichiometries were calculated from the decrease in fluorescence of residue -Trp-365. The solid line represents a fit to the data points obtained in absence of P or PP (2.8 identical sites with K = 16 µM), the dotted and dashed lines are fits to the data points for 100 and 200 µM PP, respectively.



Therefore several points have been established. First, MgAMP-PNP binds to all three F noncatalytic sites, in agreement with previous work which utilized radioactive MgAMP-PNP (Wise et al., 1983), and similar to MgATP and MgADP (Weber et al., 1994b). Second, the presence of 5 mM P had zero effect on MgAMP-PNP binding, showing that P does not bind at noncatalytic sites. Third, it is evident that MgPP does compete for binding of MgAMP-PNP at all three noncatalytic sites. Fourth, the apparent binding affinity for MgPP at noncatalytic sites is similar to that for Mg nucleotides.

Use of a MgPP Trap to Measure Dissociation of Bound Nucleotides from Noncatalytic Sites

Nucleotide-depleted AW7 F was incubated in buffer containing 50 mM Tris-SO, 2.5 mM MgSO, pH 8.0, for 20 s in presence of 1 mM ATP or GTP. At this concentration of nucleotide, >2 noncatalytic sites were filled in the MgATP-reloaded enzyme and 1.1 in MgGTP-reloaded F (consistent with previous data of Weber et al., 1994b). The enzyme solutions were then diluted 200-fold into the same buffer containing 0.2 mM NaPP, such that final enzyme concentration was 50 nM, and the fluorescence signals were monitored. With MgGTP-reloaded F, the fluorescence signal reached immediately the same level as that of the control (preincubated without nucleotides). This means that virtually all GTP had been released during mixing time (about 15 s). Thus, we could only estimate an upper limit of 5 s for the apparent half-time for dissociation of GTP from the noncatalytic sites (k >0.1 s). In contrast, release of ATP was much slower. The apparent half-time for dissociation of ATP was 8-11 min (k = 1.0-1.4 10 s).

The experiments were repeated using a preincubation period of 1 h instead of 20 s. No difference was seen with the enzyme preloaded with MgGTP (k >0.1 s), but with MgATP the k decreased to 2-4 10 s. (Since 5 µM nucleotide was present in the medium during the dissociation phase of the experiment, these values are apparent values only, however, it should be noted that when ATP-loaded F was passed through a centrifuge column immediately before the dilution step, the same results were obtained).

Native AW7 F, in which the noncatalytic sites were already nearly fully occupied by endogenous adenine nucleotide (2.7 mol/mol F), was incubated with 1.0 mM NaPP in 50 mM Tris-SO, 2.5 mM MgSO, pH 8.0, 23 °C, and it was seen that over a period of 3 h there was no measureable release of nucleotide from the noncatalytic sites. Therefore in the native AW7 enzyme the dissociation rate of adenine nucleotide is very slow indeed. Additionally, inclusion of 50 µM ATP to induce enzyme turnover did not cause nucleotide release.

Effects of MgPP on ATP Hydrolysis

Using wild-type F we tested the effects of MgPP in assays of steady-state ATP hydrolysis. In these experiments, nucleotide-depleted enzyme was compared with native F; the latter preparation contained noncatalytic sites essentially fully loaded with adenine nucleotides (Weber et al., 1994b). Both enzymes were preincubated with 1 mM NaPP in buffer containing 50 mM Tris-SO, 2.5 mM MgSO, pH 8.0, 23 °C, then diluted into ATPase assay medium (final concentrations: 50 mM Tris-SO, pH 8.0, 1 mM PP, 10-2000 µM ATP, MgSO equal to the sum of PP and ATP concentrations). There was no significant difference in ATPase activity between the native and nucleotide-depleted enzymes. Under the conditions of the experiment, the noncatalytic sites of the nucleotide-depleted enzyme would have been occupied by MgPP, and we may conclude that this did not affect the rates of catalysis to a significant extent.


DISCUSSION

General Comments

The use of mutant E. coli F-ATPase enzymes containing tryptophans substituted specifically in catalytic sites (Y331W) or noncatalytic sites (R365W) has enabled us to determine at which of these sites PP or P compete with nucleoside triphosphate for binding, and to determine apparent K values. summarizes the data for binding of different ligands to the catalytic and noncatalytic sites, derived from work presented here and from previous reports (Weber et al., 1993a, 1994a, 1994b). The data for MgPP are shown in , line 5. It is seen that MgPP binds at noncatalytic sites, and the apparent K (MgPP) of 20 µM is surprisingly similar to that for MgAMP-PNP, MgATP, and MgADP, as measured by the tryptophan fluorescence signal. Occupancy of the noncatalytic sites by MgPP had no effect on ATP hydrolysis. Neither MgPP () nor free PP (see ``Results'') was seen to bind significantly to catalytic sites. P (in presence of Mg) did not bind to either type of site (). Further to the data in , it may be noted that MgGTP binds to both types of site whereas MgGDP binds to catalytic but not to noncatalytic sites (Weber et al., 1994a, 1994b; Hyndman et al., 1994)

Free PP caused net displacement of nucleotide from catalytic sites of native enzyme; this effect was mimicked by EDTA and is apparently related to the ability of PP to complex Mg ions, as discussed below. There was no measurable release of nucleotide from noncatalytic sites of native enzyme in presence of PP; however release of nucleotide from noncatalytic sites of enzyme which had been nucleotide-depleted then reloaded was faster. MgPP was used as a trap to allow determination of relative dissociation rates for reloaded MgGTP and MgATP. Based on the results, a model for nucleotide binding to noncatalytic sites is described below.

The Binding Process at Noncatalytic Sites

Nucleotide binding at noncatalytic sites shows anomalous features. For example, when monitored by the -Trp-365 fluorescence signal, binding of MgATP to AW7 F occurred with an apparent K = 25 µM and k = 270 M s (Weber et al., 1994b). As was discussed in that paper, this leads to a calculated k value of 6.8 10 s, which is much faster than expected from previous work in which nucleotide-depleted wild-type F was reloaded with radioactive MgATP and dissociation of radioactivity followed (Pagan and Senior, 1990), and also much faster than the dissociation of nucleotides from the noncatalytic sites of native AW7 F (this paper). We suggested in Weber et al., (1994b) that, for MgATP, subsequent to the initial binding event which is recorded by the fluorescence signal, a conformational change occurs in the noncatalytic site which has the effect of considerably reducing k. It was further proposed that guanine nucleotide is not able to bring about this conformational change, and so the apparent k for MgGTP remains relatively high.

Our finding that the K for MgPP is similar to that for MgATP, MgADP and MgAMP-PNP, when measured by the fluorescence technique () leads us to propose that in the initial phase of binding of nucleotide at noncatalytic sites it is the pyrophosphate ``end'' of the nucleotide that is recognized. The initial phase of binding is proposed to lead to ``State I'' in which the site is now occupied, but the nucleotide has a relatively fast k. Then, if the nucleotide contains adenine as the base, a conformational change is induced which sequesters the nucleotide in ``State II,'' rendering it ``non-exchangeable'' by reducing k. This conformational change does not apparently occur with guanine nucleotide. Supportive of this, using MgPP as a trap to prevent rebinding of nucleotide, we have shown here that in enzyme that had been depleted of endogenous nucleotides and then reloaded, the dissociation rate of MgGTP is much faster than that of MgATP. Also, the dissociation rate measured for MgATP reloaded into nucleotide-depleted F was lower than that calculated from the K (MgATP) (above). Prolonging the MgATP ``reloading period'' decreased the dissociation rate further. Still, however, it was considerably higher than that for dissociation of endogenous nucleotides (usually a mixture of ADP and ATP; see Wise et al., 1981; Issartel et al., 1986; Senior et al., 1992) from native F. Thus it appears as if there are additional factors involved in the native enzyme that decrease nucleotide dissociation rates from noncatalytic sites.

Functional Comparisons between Catalytic and Noncatalytic Sites

In both catalytic and noncatalytic nucleotide-binding sites of F, residues from the ``Homology A'' and ``Homology B'' consensus sequences play a prominent role in binding the phosphate moieties of the nucleotides (Abrahams et al. 1994). One difference between the structures in the two types of site that might explain the presence of catalytic activity in the former but not in the latter has been discussed by Abrahams et al., who suggested that residue -Glu-181 of the catalytic site may be critical. This suggestion is fully consistent with earlier kinetic and thermodynamic analyses of catalytic properties of the E181Q mutant enzyme (Senior and Al-Shawi, 1992). The equivalent residue in the noncatalytic site is -Gln-208. A second difference between the two types of site is that the noncatalytic sites are relatively specific for adenine nucleotide (Perlin et al., 1984; Weber et al., 1994b) in comparison to the catalytic sites. Abrahams et al.(1994) suggest that this could be due to hydrogen-bonding properties of the residues surrounding the base moiety of the nucleotide in the two types of site. The dissociation rates for MgGTP versus MgATP (above) provide experimental explanation for this apparent specificity of the noncatalytic sites. A third difference is that the catalytic sites bind nucleotides not only in the presence but also in the absence of Mg, albeit with altered characteristics; in contrast, the noncatalytic sites are strongly Mg-dependent (Weber et al., 1994a, 1994b). The work presented here defines a fourth, clear-cut, difference between the catalytic site and the noncatalytic site, namely that significant binding of MgPP is seen only in the latter. A structural rationale for this difference is not yet known, and its elucidation will be of considerable interest. From this work it is apparent that measurements of MgPP binding parameters in wild-type and particularly mutant E. coli enzymes can now be used as a distinguishing functional characteristic of the noncatalytic sites. We anticipate that this will facilitate studies of the physiological role of the noncatalytic sites which, despite much research, remains obscure.

Displacement of Nucleotides from the Catalytic Sites by PP Is Due to Complexation of Mg Ions

We confirmed here previous results showing that PP was apparently able to displace nucleotide from the catalytic sites, and yet we found that neither free PP nor MgPP was able to bind to catalytic sites. The explanation for this conundrum appears to reside in the fact that PP complexes with Mg ions relatively strongly. In a solution of native F, MgADP will be continually released from the catalytic sites of the enzyme into the medium. Whereas this nucleotide could normally rebind readily as MgADP, in the presence of an excess of PP some of the MgADP will lose Mg to PP, and consequently its rebinding will be impaired. Thus the apparently conflicting data in the literature are satisfactorily resolved.

Filling of the Noncatalytic Sites with MgPP Has No Effect on Catalysis

In this study we demonstrate that MgPP binding at noncatalytic sites had no effect on ATP hydrolysis in E. coli F. Jault and Allison (1993) have suggested that the effect of PP to increase the ATPase activity in bovine mitochondrial F-ATPase is caused by the fact that binding of PP into the three noncatalytic sites induces release of long-lived inhibitory MgADP from a catalytic site. In a previous study we demonstrated that long-lived MgADP inhibition is not found in E. coli F as prepared here (Senior et al., 1992). Therefore the lack of an effect of PP in E. coli F is not unexpected. Given that MgPP did not displace adenine nucleotide from the noncatalytic sites of native F (above) and taking into account the ambient cytoplasmic ATP concentrations of 2-3 mM in E. coli cells, it is not likely that MgPP plays a role in physiological regulation of FF. The major value of this study therefore is to show that PP can be a valuable experimental tool for stucture-function studies of the nucleotide sites.

  
Table: Binding of MgAMP-PNP to the catalytic sites in absence and presence of MgPP

Binding parameters for MgAMP-PNP were obtained by fitting theoretical curves to the data shown in Fig. 2. A good fit was obtained assuming two types of binding sites with N = 1.0 and N = 1.5, both in absence and presence of MgPP. Dissociation constants are given ± standard deviation.


  
Table: Binding of ligands to nucleotide sites in E. coli F as determined by fluorescence signal of W365 or W331 residues



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM25349 (to A. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Unless noted otherwise, the residue numbers given refer to the E. coli enzyme.

The abbreviation used is: AMP-PNP, adenyl-5`-yl imidodiphosphate.

The residual signal is probably due to contaminating proteins in the CB5 F preparation. Quantification of the total tryptophan content after denaturation in 6 M guanidine hydrochloride gave a value of 4.0 mol/mol F, rather than 3 mol/mol as expected. This means that the protein contamination would amount to approximately 2% if an average tryptophan content is assumed (see Wilke-Mounts et al. (1994)).


ACKNOWLEDGEMENTS

We thank Cheryl Bowman for excellent technical assistance.


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