Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF<Subscript>1</Subscript>

This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more.     

Evaluation by steady-state enzyme kinetics of the role of tightly bound nucleotides during photophosphorylation

The ATP synthetase of chloroplast membranes binds ADP and ATP with high affinity, and the binding becomes quasi-irreversible under certain conditions. One explanation of the function of these nucleotides is that they are transiently tightly bound during ATP synthesis as part of the catalytic process, and that the release of tightly bound ATP from one catalytic site is promoted when ADP and P(i) bind to a second catalytic site on the enzyme. Alternatively, it is possible that the tightly bound nucleotides are not catalytic, but instead have some regulatory function. We developed steady-state rate equations for both these models for photophosphorylation and tested them with experiments where two alternative substrates, ADP and GDP, were phosphorylated simultaneously. It was impossible to fit the results to the equations that assumed a catalytic role for tightly bound nucleotides, whether we assumed that both ADP and GDP, or only ADP, are phosphorylated by a mechanism involving substrate-induced release of product from another catalytic site. On the other hand, the equations derived from the regulatory-site model that we tested were able to fit all the results relatively well and in an internally consistent manner. We therefore conclude that the tightly bound nucleotides most likely do not derive from catalytic intermediates of ATP synthesis, but that substrate (and possibly also product) probably bind both to catalytic sites and to noncatalytic sites. The latter may modulate the transition of the ATP-synthesizing enzyme complex between its active and inactive states.     

Regulation of Escherichia coli glutamine synthetase. Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme.

The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques. These studies show that two manganese ions bind per enzyme subunit. From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites. Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion. The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site. This suggests that alanine binds closer to the more tightly bound manganese ion. Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion. It is proposed that alanine and glutamine bind competitively and in the same site. The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand. The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present. Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate. A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.     

Binding of adenine nucleotides to the F1-inhibitor protein complex of bovine heart submitochondrial particles.

The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase

The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked inhibition.     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＾＋－ＡＴＰ酶复合中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ时,Ｆｏ的荧光猝灭常数的变化结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝来常数,也就是Ｆｏ会产生不同的构象变化。这些结果说明了Ｈ＾＋ＡＴＰ酶合ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＋－ＡＴＰ酶复合体（也称ＡＴＰ合成酶）中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ（酶蛋白与底物分子比为１：１）时,Ｆｏ的荧光猝灭常数的变化（用竹红菌乙作为膜区蛋白荧光的猝灭剂）结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝灭常数（Ｋｓｖ）,也就是Ｆｏ会产生不同的构象变化。加入二价金属离子起动ＡＴＰ水解反应结束后：ＡＴＰ＋Ｈ２Ｏ→ＡＤＰ＋Ｐｉ,这时可以在Ｆｏ观察到与ＡＤＰ加Ｍｇ２＋时相同猝灭常数Ｋｓｖ;用荧光强度随时间进程变化的实验可观察到Ｆ１水解过程中导致Ｆｏ构象变化的动力学过程。这些结果说明了Ｈ＋－ＡＴＰ酶复合体ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

Chloroplast F1 ATPase has more than three nucleotide binding sites, and 2-azido-ADP or 2-azido-ATP at both catalytic and noncatalytic sites labels the beta subunit

The photolabeling of chloroplast F1 ATPase, following exposure to Mg2+ and 2-azido-ATP and separation from medium nucleotides, results in derivatization of two separate peptide regions of the beta subunit. Up to 3 mol of the analogue can be incorporated per mole of CF1, with covalent binding of one moiety or two moieties per beta subunit that can be either AMP, ADP, or ATP derivatives. These results, the demonstration of noncovalent tight binding of at least four [3H]adenine nucleotides to the enzyme and the presence of three beta subunits per enzyme, point to six potential adenine nucleotide binding sites per molecule. The tightly bound 2-azido nucleotides on CF1, found after exposure of the heat-activated and EDTA-treated enzyme to Mg2+ and 2-azido-ATP, differ in their ease of replacement during subsequent hydrolysis of ATP. Some of the bound nucleotides are not readily replaced during catalytic turnover and covalently label one peptide region of the beta subunit. They are on noncatalytic sites. Other tightly bound nucleotides are readily replaced during catalytic turnover and label another peptide region of the beta subunit. They are at catalytic sites. No alpha-subunit labeling is detected upon photolysis of the bound 2-azido nucleotides. However, one or both of the sites could be at an alpha-beta-subunit interface with the 2-azido region close to the beta subunit, or both binding sites may be largely or entirely on the beta subunit.     

Binding of regulatory ligands to rabbit muscle phosphofructokinase. A model for nucleotide binding as a function of temperature and pH.

The binding of nucleoside triphosphates to rabbit muscle phosphofructokinase has been determined in 0.05 M phosphate buffers by changes in intrinsic protein fluorescence and by direct binding measurements. These experiments have been performed over a wide range of pH, temperature, and effector concentration. Quenching of protein fluorescence is shown to measure binding of nucleotides to a site which is not the active site but rather a site responsible for inhibition of the kinetic activity. This site is relatively specific for either ATP or MgATP with free ATP binding about 10-fold more tightly than MgATP. A model to describe binding to this site as a function of pH and temperature is proposed. This model assumes that the apparent affinity for ATP is determined by protonation of two ionizable groups (per subunit) and that ATP binds exclusively to protonated enzyme forms. Several ligands which affect the apparent affinity for nucleotide binding at the inhibitory site act by shifting the apparent pK of the ionizable groups. NH4+ and citrate do not influence nucleotide binding to the inhibitory site. At pH 6.9 in 0.05 M phosphate, low concentrations of MgATP or MgGTP enhance the protein fluorescence due to binding at the active site. The fluorescence studies and direct binding studies show that there is one active site and one inhibitory site per subunit. As described elsewhere (Pettigrew, D. W., and Frieden, C. (1978) J. Biol. Chem. 253, 3623-3627), there is a third nucleotide binding site on each subunit which is specific for cAMP, AMP, and ADP.     

Adenine nucleotide binding sites in normal and mutant adenosine triphosphatases of Escherichia coli

Three types of assays were used to characterize adenine nucleotide binding sites on the Ca²⁺, Mg²⁺-activated ATPase of normal Escherichia coli and its unc A 401 and unc D 412 mutants. ADP was bound mainly at a single site in normal and mutant ATPase. In the absence of divalent cations ATP was bound at a single high-affinity and three low-affinity sites in normal and unc D ATPases. The 2′,3′-dialdehyde (oADP) obtained by periodate oxidation of ADP reacted with both low- and high-affinity sites whereas oATP was bound primarily at a low-affinity site. Two types of adenine nucleotide binding sites, a high-affinity site reacting with ATP and ADP and a low-affinity site for ATP, were detected by the effects of these nucleotides on the fluorescence of the aurovertin D-ATPase complex. This high-affinity site(s) was present in normal and mutant ATPases. However, the fluorescence response at both high- and low-affinity sites was modified in the unc D ATPase as a consequence of the abnormal β subunit in this enzyme. Normal fluorescence responses were not induced by the binding of oADP or oATP to the ATPases. ATP was bound at a single site on isolated α subunits of the enzyme. Since this site was not detected in the unc A ATPase, it is unlikely to be the high-affinity site detected in the intact enzyme or the binding site for the endogenous tightly bound adenine nucleotides found in the purified ATPase. It is more probable that the site detected on the isolated α subunit from the normal enzyme is that which binds oADP since this site was absent in the unc A ATPase. Pretreatment of the normal ATPase with either N, N′-dicyclohexyl-carbodiimide (DCCD) or with 4-chloro-7-nitrobenzofurazan (NbfCl), reagents which inhibit ATPase activity by reacting with a β subunit, affected binding of oADP to α subunit(s) but had less effect with oATP. Inhibition of oADP binding could be due to conformational changes induced in the α subunit by the reaction of DCCD and NbfCl with a β subunit, or to steric reasons. If the latter hypothesis is correct, the active site of the ATPase would be at the interface between α and β subunits of the enzyme.     

Effects of the chaperonin GroE on the refolding of tryptophanase from Escherichia coli. Refolding is enhanced in the presence of ADP.

The refolding of the tetrameric enzyme tryptophanase was facilitated by the chaperonin GroE. Maximum refolding yield of tryptophanase molecules (about 80%) was attained in the presence of a 15-fold excess of GroE 21-mer over tryptophanase monomer. The GroEL subunit was required for this improvement in refolding yield, whereas the GroES subunit was not. Light scattering experiments of the refolding reaction revealed that GroE bound to tryptophanase folding intermediates and suppressed their aggregation. The presence of ATP was required for the efficient dissociation of tryptophanase from GroEL. However, our experiments indicated that tryptophanase dissociated readily from GroEL in the presence of not only ATP, but also in the presence of non-hydrolyzable ATP analogues such as ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) and AMP-PNP (adenyl-5'-yl imidodiphosphate) as well. Surprisingly, the release of tryptophanase from GroEL was facilitated in the presence of ADP as well. We concluded that the binding of nucleotides such as ATP and ADP changed the conformation of GroEL and facilitated the dissociation of tryptophanase molecules. The conformation formed in the presence of ADP was distinct from the conformation formed in the presence of ATP, as shown by the selective dissociation of various folding proteins from the two conformations.     

Inhibition of mitochondrial F1-ATPase activity by binding of (2-azido-) ADP to a slowly exchangeable non-catalytic nucleotide binding site.

F1-ATPase was treated so that it contained three tightly bound nucleotides per molecule. One of these was bound at a catalytic site and was rapidly exchangeable, the two remaining nucleotides were nonexchangeable. Incubation of this preparation with ADP in the presence of Mg2+ results in 40-45% inhibition of the ATPase activity. With 2-azido-ADP instead of ADP, the ligand was covalently bound to F1 by illumination, in the presence or absence of turnover of the enzyme, and the site of binding was determined. In this way, one site could be identified, which induces the inhibition. The attachment of the covalently bound 2-nitreno-ADP is at Tyr-368 of a beta-subunit, characterized in the literature as a non-catalytic site. A second, non-catalytic site also binds 2-azido-ADP, but this binding is partially reversed by the addition of ATP and does not cause further inhibition of the ATPase activity. It is concluded that the slowly exchangeable non-catalytic site is the site of inhibition by ADP.     

Modulation of the chloroplast ATPase by tight binding of nucleotides

Inactivation of the chloroplast ATPase upon tight nucleotide binding was studied with several adenine nucleotide analogs. Compared with ADP, the other nucleoside diphosphates were less effective in the follwing order: IDP >?-ADP > 1-oxido-ADP > GDP. The nucleotide analogs compete with ADP for binding to the tight nucleotide-binding site(s) on the ATPase and also prevent further inactivation by ADP. AdoPP[NH]P also causes inactivation but has a lower affinity than ADP. [³H]GDP binds tightly to the ATPase, but the resulting enzyme-GDP complex is more readily dissociable than the enzyme-ADP complex. Although both nucleotides appear to bind to the same site, the catalytic and binding properties of the coresponding nucletide-enzyme complexes differ. Binding of GDP also decreases the rate and extent of the sontaneous decay of the activated enzyme. PP_i decreases the rate of inacivation caused by ADP and also the level of tigthly buond ADP. Based on these results, we suggest that two different confomations of the ATPase exist which contain tigthly bound ADP. The active conformation is conveted to the inactive conformation in the absence of a continued supply of energy by illumination or ATP hydrolysis.     

The catalytic site is located on subunit I of the ATPase from Halobacterium saccharovorum. A direct photoaffinity labeling study.

Nucleotide-binding sites of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum were labeled by ultraviolet irradiation in the presence of [alpha-32P]ATP. A high-affinity site, located on subunit I (98 kDa), was identified as catalytic by the following criteria: ATP bound to subunit I was hydrolyzed and the cross-linked nucleotide was ADP; the specificity for ATP or ADP compared to that of other nucleotides was high; the tightly bound radionucleotide was exchangeable in the presence of excess unlabeled ATP and Mg2+; photolabeling of this site and enzyme inhibition due to tightly bound ADP were both dependent on the presence of Mg2+ and showed identical Kd values; treatment that restored the activity of the ADP-inhibited enzyme also led to the release of the tightly bound nucleotide from subunit I. In addition, a non-catalytic nucleotide-binding site was found, located on subunit II (71 kDa). This site did not hydrolyze ATP, its occupation was Mg2+ independent and the affinity for ATP and the nucleotide specificity were much lower than that of subunit I. We suspect that this site is nonspecific. These results indicate that H. saccharovorum ATPase is different from F1-ATPases which contain the catalytic site on the second largest subunit, but may be similar to other archaebacterial and vacuolar ATPases.     

Nucleotide binding sites and chemical modification of the chromaffin granule proton ATPase

The purified proton ATPase of chromaffin granules contains five different polypeptides denoted as subunits I to V in the order of decreasing molecular weights of 115,000, 72,000, 57,000, 39,000, and 17,000, respectively. The purified enzyme was reconstituted as a highly active proton pump, and the binding of N-ethylmaleimide and nucleotides to individual subunits was studied. N-Ethylmaleimide binds to subunits I, II, and IV, but inhibition of both ATPase and proton pumping activity correlated with binding to subunit II. In the presence of ADP, the saturation curve of ATP changed from hyperbolic to a sigmoid shape, suggesting that the proton ATPase is an allosteric enzyme. Upon illumination of the purified enzyme in the presence of micromolar concentrations of 8-azido-ATP, alpha-[35S]ATP, or alpha-[32P]ATP subunits I, II, and IV were labeled. However, at concentrations of alpha-[32P]ATP below 0.1 microM, subunit II was exclusively labeled in both the purified and reconstituted enzyme. This labeling was absolutely dependent on the presence of divalent cations, like Mg2+ and Mn2+, while Ca2+, Co2+, and Zn2+ had little or no effect. About 0.2 mM Mg2+ was required to saturate the reaction even in the presence of 50 nM alpha-[32P]ATP, suggesting a specific and separate Mg2+ binding site on the enzyme. Nitrate, sulfate, and thiocyanate at 100 mM or N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at 100 microM prevented the binding of the nucleotide to subunit II. The labeling of this subunit was effectively prevented by micromolar concentrations of three phosphonucleotides including those that cannot serve as substrate for the enzyme. It is concluded that a tightly bound ADP on subunit II is necessary for the activity of the enzyme.     

Four tight nucleotide binding sites of chloroplast coupling factor 1.

We have examined the properties of the four tight nucleotide binding sites of reductively activated chloroplast coupling factor 1. Tight sites are here defined as those which retain bound nucleotides after passage of the chloroplast coupling factor 1 through Sephadex gel filtration centrifuge columns. Two of the sites, here called sites 4 and 5, have not been characterized in detail before. Site 4 has properties similar to those of site 1. It binds to ADP, ATP, and adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) tightly in the presence or absence of Mg2+. Bound ADP exchanges rapidly with medium ADP, but rapid exchange with ATP or AMP-PNP requires Mg2+. Site 4 may slowly hydrolyze bound ATP in the absence of medium nucleotides. Site 5 has properties similar to those of site 2. Tight binding of ATP and AMP-PNP requires Mg2+, but Mg29+)-ADP is not tightly bound. Site 5 does not hydrolyze bound ATP in the absence of medium nucleotides. Complete filling of all four tight nucleotide binding sites requires about one millimolar nucleotide, suggesting that low affinity binding sites are converted to tight binding via a nucleotide binding-induced conformational change.     

Loose and tight binding of adenine nucleotides by membrane-associated chloroplast ATPase

Steady-state binding of adenine nucleotides by thylakoid membranes is measured by employing a centrifugation technique. By this method tightly bound nonexchangeable nucleotides can be discriminated from loosely bound, exchangeable nucleotides. Nucleotide binding requires membrane energization and is highly specific for medium ADP. In illuminated chloroplasts almost no exogenous AMP and only some ATP are incorporated, most being recovered as tightly bound nucleotides. In light-triggered chloroplasts, however, which are capable of hydrolyzing ATP, a high level of exchangeable nucleotides is found on the membranes. The sum of tightly bound and loosely bound nucleotides originating from medium ADP is about one per CF₁. The ratio between them decreases with increasing proton-motive force. Exchangeable nucleotides most probably represent the ligands involved in the catalytic process, as suggested from substrate specificity and the effect of a competitive inhibitor of photophosphorylation, naphthoyl ADP. This compound in a low concentration range supresses loose binding but not tight binding of medium ADP. Under phosphorylating conditions (presence of ADP, P_i and light), some of the tightly bound nucleotides exist as ATP even in the presence of a hexokinase system. The results are discussed in the context of the regulation of chloroplast ATPase by tight nucleotide binding.     

Function of tightly bound nucleotides on membrane-bound chloroplast coupling factor

The kinetic behavior of tightly bound nucleotides on chloroplast coupling factor from spinach was determined under phosphorylating and nonphosphorylating conditions. Chloroplast coupling factor 1 (CF1) was labeled with tightly bound radioactive ADP and/or ATP at two specific sites and reconstituted with thylakoid membranes depleted of CF1 by treatment with NaBr. The initial incorporation and dissociation of ADP from one of the sites requires light but occurs at the same rate under phosphorylating and non-phosphorylating conditions. The initial rate is considerably slower than the rate of ATP synthesis, but nucleotide exchange is very rapid during steady-state ATP synthesis. A direct correspondence between this nucleotide binding site and a site on soluble CF1 that hydrolyzes ATP was demonstrated. A second site binds MgATP very tightly; the MgATP does not dissociate during ATP synthesis nor does its presence alter the rate of ATP synthesis. This is analogous to the behavior found for soluble CF1 during ATP hydrolysis. These results demonstrate that the tight-binding nucleotide sites on soluble CF1 and membrane-bound coupling factor are essentially identical in terms of binding properties and kinetic behavior during ATP hydrolysis and synthesis.     

Characterization of exchangeable and nonexchangeable bound adenine nucleotides in F1-ATPase from Escherichia coli

The total amount of bound exchangeable and nonexchangeable adenine nucleotides in Escherichia coli F1-ATPase (BF1) was determined; three exchangeable nucleotides were assessed by equilibrium dialysis in a [14C]ADP-supplemented medium. When BF1 was purified in a medium supplemented with ATP, a stoichiometry of nearly 6 mol of bound nucleotides/mol of enzyme was found; three of the bound nucleotides were ATP and the others ADP. When BF1 was filtered on Sephadex G-50 in a glycerol medium (Garrett, N.E., and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647), bound ADP was rapidly released, in contrast to bound ATP which remained firmly attached to the enzyme. Upon incubation of BF1 with [14C]ADP, the bound ADP rather than the bound ATP was exchanged. Of the three [14C]ADPs which have bound to BF1 by exchange after equilibrium dialysis, one was readily lost by gel filtration on Sephadex G-50; the loss of bound [14C]ADP was markedly reduced by incubation of BF1 with aurovertin, a specific ligand of the beta subunit which is known to increase the affinity of the beta subunit for nucleotides (Issartel, J.-P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). Upon photoirradiation of BF1 with [alpha-32P]2-azido-ADP, only the beta subunit was labeled; concomitantly, bound ADP was released, but the content in bound ATP remained stable. These results suggest that specific sites located on the three beta subunits bind nucleotides in a reversible manner. Consequently, the tightly bound ATP of native BF1 would be located on the alpha subunits.     

Further characterization of nucleotide binding sites on chloroplast coupling factor one

The solubilized coupling factor from spinach chloroplasts (CF1) contains one nondissociable ADP/CF1 which exchanges slowly with medium ADP in the presence of Ca2+, Mg2+, or EDTA; medium ATP also exchanges in the presence of Ca2+ or EDTA, but it is hydrolyzed, and only ADP is found bound to CF1. The rate of ATP exchange with heat-activated CF1 is approximately 1000 times slower than the rate of ATP hydrolysis. In the presence of Mg2+, both latent CF1 and heat-activated CF1 bind one ATP/CF1, in addition to the ADP. This MgATP is not removed by dialysis, by gel filtration, or by the substrate CaATP during catalytic turnover; however, it is released when the enzyme is stored several days as an ammonium sulfate precipitate. The photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]-propionyl]-ATP binds to the MgATP site, and photolysis results in labeling of the beta subunit of CF1. Equilibrium binding measurements indicate that CF1 has two identical binding sites for ADP with a dissociation constant of 3.9 microM (in addition to the nondissociable ADP site). When MgATP is bound to CF1, one ADP binding site with a dissociation constant of 2.9 microM is found. One ATP binding site is found in addition to the MgATP site with a dissociation constant of 2.9 microM. Reaction of CF1 with the photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ADP indicates that the ADP binding site which is not blocked by MgATP is located near the interface of alpha and beta subunits. No additional binding sites with dissociation constants less than 200 micro M are observed for MgATP with latent CF1 and for CaADP with heat-activated CF1. Thus, three distinct nucleotide binding sites can be identified on CF1, and the tightly bound ADP and MgATP are not at the catalytic site. The active site is either the third ADP and ATP binding site or a site not yet detected.