The role of tightly bound ADP on chloroplast ATPase

Isolated chloroplast coupling factor 1 ATPase is known to retain about 1 mol of tightly bound ADP/mol of enzyme. Some experimental results have given evidence that the bound ADP is at catalytic sites, but this view has not been supported by observations of a slow replacement of the bound ADP when CaATP or MgATP is added. The experiments reported in this paper show why a slow replacement of ADP bound at a catalytic site can occur. When coupling factor 1, labeled with tightly bound [3H]ADP, is exposed to Mg2+ or Ca2+ prior to the addition of MgATP or CaATP, a pronounced lag in the onset of ATP hydrolysis is observed, and only slow replacement of the [3H]ADP occurs. Mg2+ or Ca2+ can induce inhibition very rapidly, as if an inhibited form of the enzyme results whenever the enzyme with tightly bound ADP encounters Mg2+ or Ca2+ prior to ATP. The inhibited form can be slowly reactivated by incubation with EDTA, although some irreversible loss in activity is encountered. In contrast, when MgATP or CaATP is added to enzyme depleted of Mg2+ and Ca2+ by incubation with EDTA, a rapid onset of ATP hydrolysis occurs and most of the tightly bound [3H]ADP is released within a few seconds, as expected for binding at a catalytic site. The Mg2+-induced inhibition of both the ATPase activity and the lack of replacement of tightly bound [3H] ADP can be largely prevented by incubation with Pi under conditions favoring Pi addition to the site containing the tightly bound ADP. Our and other results can be explained if enzyme catalysis is greatly hindered when MgADP or CaADP without accompanying Pi is tightly bound at one of the three catalytic sites on the enzyme in a high affinity conformation.     

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.     

Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.     

Significant quantities of endogenous GDP and ADP are present on catalytic sites of the F1-ATPase isolated from M. lysodeikticus in the absence of added nucleotides.

The F1-ATPase from Micrococcus lysodeikticus is isolated in the absence of exogenous nucleotides. After removing loosely bound nucleotides from the isolated enzyme by gel permeation chromatography, analysis for tightly bound nucleotides revealed in 14 experiments 0.4 +/- 0.1 mol ADP, 0.5 +/- 0.2 mol GDP, and 0.8 +/- 0.2 mol ATP per mol of F1. Incubation of the isolated enzyme with Mg2+ or Ca2+ did not alter the endogenous nucleotide composition of the enzyme, indicating that endogenous ATP is not bound to a catalytic site. Incubation of the enzyme with P(i) decreased the amount of tightly bound ADP and GDP but did not effect the ATP content. Hydrolysis of MgATP in the presence of sulfite raised the tightly bound ADP and lowered tightly bound GDP on the enzyme. In the reciprocal experiment, hydrolysis of MgGTP in the presence of sulfite raised tightly bound GDP and lowered tightly bound ADP. Turnover did not affect the content of tightly bound ATP on the enzyme. These results suggest that endogenous ADP and GDP are bound to exchangeable catalytic sites, whereas endogenous ATP is bound to noncatalytic sites which do not exchange. The presence of endogenous GDP on catalytic sites of isolated F1 suggests that the F0F1-ATP synthase of M. lysodeikticus might synthesize both GTP and ATP under physiological conditions. In support of this hypothesis, we have found that plasma membrane vesicles derived from M. lysodeikticus synthesize [32P]GTP from [32P]P(i) using malate as electron donor for oxidative phosphorylation.     

Active/Inactive state transitions of the chloroplast F1 ATPase are induced by a slow binding and release of Mg2+. Relationship to catalysis and control of F1 ATPases

Mg2+ is known to be a potent inhibitor of F1 ATPases from various sources. Such inhibition requires the presence of a tightly bound ADP at a catalytic site. Results with the spinach chloroplast F1 ATPase (CF1) show that the time delays of up to 1 min or more in the induction or the relief of the inhibition are best explained by a slow binding and slow release of Mg2+ rather than by slow enzyme conformational changes. CF1 is known to have multiple Mg2+ binding sites with Kd values in the micromolar range. The inhibitory Mg2+ and ADP can bind independently to CF1. When Mg2+ and ATP are added to the uninhibited enzyme, a relatively fast rate of hydrolysis attained soon after the addition is followed by a much slower steady-state rate. The inhibited steady-state rate results from a slowly attained equilibrium of binding of medium Mg2+. The Kd for the binding of the inhibitory Mg2+ is in the range of 1-8 microM, in the presence or absence of added ATP, as based on the extent of rate inhibition induced by Mg2+. Assessments from 18O exchange experiments show that the binding of Mg2+ is accompanied by a relatively rapid change to an enzyme form that is incapable of hydrolyzing MgATP. When ATP is added to the Mg2+- and ADP-inhibited enzyme, the resulting reactivation can be explained by MgATP binding to an alternate catalytic site which results in a displacement of the tightly bound ADP after a slow release of Mg2+. Both an increase in temperature (to 50 degrees C) and the presence of activating anions such as bicarbonate or sulfite reduce the extent of the Mg2+ inhibition markedly. The activating anions may bind to CF1 in place of Pi near the ADP. Whether the inhibitory Mg2+ binds at catalytic or noncatalytic nucleotide binding sites or at another location is not known. The Mg2(+)- and ADP-induced inhibition appears to be a general property of F1 ATPases, which show considerable differences in affinity for ADP, Mg2+, and Pi. These differences may reflect physiological control functions.     

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.     

Mitochondrial F1-ATPase will bind and cleave ATP but only slowly release ADP after N,N'-dicyclohexylcarbodiimide or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatization

The ATPase from the inner mitochondrial membrane is known to be inhibited by modification of one of the three catalytic subunits with N,N'-dicyclohexylcarbodiimide (DCCD) or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. An experimental approach described in this paper shows that most of the residual ATPase activity observed after the usual DCCD or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole modification is due to the presence of unmodified enzyme, although the large fraction of modified enzyme retains a weak catalytic activity. This weak catalytic activity can be stimulated by methanol or dimethyl sulfoxide. When the modified enzymes are exposed to Mg2+ and [3H]ATP, about equal amounts of [3H]ATP and [3H]ADP appear at catalytic sites. The turnover rate for these enzymes is less than 1/1000 that of the native enzyme when it is calculated from the rate at which the enzyme becomes labeled at the catalytic sites with [3H]ATP and [3H]ADP during steady state hydrolysis. In addition, a higher ATP concentration is required for steady state turnover and, after ATP binding, the principal rate-limiting step is the capacity of the derivatized enzyme to undergo the binding changes necessary for the release of ADP and Pi. When the modified enzymes are not hydrolyzing ATP, they convert to form(s) that show a distinct lag in the replacement of bound nucleotides at catalytic sites. The replacement of bound nucleotides is still promoted by MgATP, even though the enzymes have been converted to sluggish forms. Contrary to a recent suggestion based on the study of the DCCD-modified enzyme (Soong, K.S., and Wang, J.H. (1984) Biochemistry 23, 136-141), our data provide evidence for the existence of catalytic cooperatively between at least two alternating sites in the modified enzyme and are consistent with continued sequential participation of all three sites.     

The mechanism of stimulation of MgATPase activity of chloroplast F1-ATPase by non-catalytic adenine-nucleotide binding. Acceleration of the ATP-dependent release of inhibitory ADP from a catalytic site.

The presence of ATP at non-catalytic sites of the chloroplast F1-ATPase (CF1) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266, 11551-11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF1. Results reported here show that when assayed in the presence of sulfite, CF1, with some non-catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non-catalytic sites is not necessary for catalytic turnover of CF1. However, even though CF1 with empty non-catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non-catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF1 results from the presence of sulfite, with or without nucleotide binding at non-catalytic sites, the Km remains constant, at about 50 microM for MgATP and 400 microM for MgGTP. The results obtained show that the ATPase activity of CF1 is determined by the fraction of the active enzyme. The inactive CF1.ADP.Mg2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non-catalytic sites promotes the ATP-dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non-catalytic sites results from increasing the portion of CF1 in an active state during steady-state ATP hydrolysis.     

Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP

The interaction of protein substrates with protease La from Escherichia coli enhances its ability to hydrolyze ATP and peptide bonds. These studies were undertaken to clarify how unfolded proteins allosterically stimulate this ATPase activity. The tetrameric protease can bind four molecules of ATP, which activates proteolysis, or four molecules of ADP, which inhibits enzymatic activity. Protein substrates stimulate binding of the nonhydrolyzable ATP analog [3H] adenyl-5'yl imidodiphosphate, although they do not increase the net binding of [3H]ATP or [3H]ADP. Once bound, ATP is quickly hydrolyzed to ADP, which remains noncovalently associated with protease La even through repeated gel filtrations. Exposure to protein substrates (e.g. denatured bovine serum albumin at 37 degrees C) induces the release of all the bound ADP from the enzyme. Nonhydrolyzable ATP analogs bound to the enzyme were not released by these substrates. Proteins that are not degraded (e.g. native bovine serum albumin) and oligopeptides that only bind to the catalytic site do not induce ADP release. Thus, polypeptide substrates have to interact with an allosteric site to induce this effect. The protein-induced ADP release is inhibited by high concentrations of Mg2+ and is highly temperature-dependent. Protein substrates promoted [3H]ATP binding in the presence of ADP and Mg2+ (i.e. ATP-ADP exchange) and reduced the ability of ADP to inhibit the enzyme's peptidase and ATPase activities. These results indicate that: 1) ADP release is a rate-limiting step in protease La function; 2) bound ADP molecules inhibit protein and ATP hydrolysis in vivo; 3) denatured proteins interact with the enzyme's regulatory site and promote ADP release, ATP binding, and their own hydrolysis.     

The substrate proton of the pyruvate kinase reaction

The pyruvate kinase reaction occurs in separate phosphate- and proton-transfer stages: (formula; see text) K+, Mg2+, and Mg.ADP are known to be required for the phosphoryl transfer step, and K+ and Mg2+ with allosteric stimulation by MgATP are important for proton transfer. This paper uses the isotope trapping method with 3H-labeled water to identify the proton donor and determine when in the sequence of the catalytic cycle it is generated. When the enzyme was allowed to exchange briefly with 3H2O (pulse phase) and then diluted into a mixture containing PEP, ADP, and the cofactor K+, Mg2+, or Co2+ in D2O (chase phase), an amount of [3H]pyruvate was formed in great excess of the amount expected from steady-state catalysis in the diluted 3H-labeled water. With K+, Mg2+, and ADP at pH 6-9.5 in the pulse phase, a limit of 1.25 enzyme equiv of 3H were trapped. The concentration of PEP required for half-maximum trapping was 14-fold greater than its steady-state Km. Therefore, the rate constant for dissociation of the donor proton is estimated to be 14 times the steady-state rate of [3H]pyruvate formation, approximately 109 s-1, or 1500 s-1. At pD 6.4, Mg2+ and ADP were required in the chase, indicating that the ADP in the pulse was not bound tightly enough to be used in the chase. At pD 9.4, ADP was not required in the chase, only Mg2+ or Co2+, making it possible to limit the chase to one turnover from hybrid labeled complexes such as E.K.Mg.CoADP or E.K.Co.MgADP and PEP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 nucleotide binding at a noncatalytic site of mitochondrial F1-ATPase accelerates a Mg(2+)- and ADP-dependent inactivation during ATP hydrolysis.

The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H-NMR studies on nucleotide binding to the sarcoplasmic reticulum Ca2+ ATPase. Determination of the conformations of bound nucleotides by the measurement of proton-proton transferred nuclear Overhauser enhancements

The glycosidic bond torsion angles and the conformations of the ribose of Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P (magnesium adenosine 5'-[beta, gamma-imido]triphosphate) bound to Ca2+ATPase, both native and modified with fluorescein isothiocyanate (FITC), in intact sarcoplasmic reticulum have been determined by the measurement of proton-proton transferred nuclear Overhauser enhancements by 1H-NMR spectroscopy. This method shows clearly the existence of a low-affinity ATP binding site after modification of the high-affinity site with FITC. For all three nucleotides bound to both the high-affinity (catalytic) site and the low-affinity site, we find that the conformation about the glycosidic bond is anti, the conformation of the ribose 3'-endo of the N type and the conformation about the ribose C4'-C5' bond either gauche-trans or trans-gauche. The values for the glycosidic bond torsion angles chi (O4'-C1'-N9-C4) for Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P bound to the low-affinity site of FITC-modified Ca2+ATPase are approximately equal to 270 degrees, approximately equal to 260 degrees and approximately equal to 240 degrees respectively. In the case of the nucleotides bound to the high-affinity (catalytic) site of native Ca2+ATPase, chi lies in the range 240-280 degrees.     

The beta G156C substitution in the F1-ATPase from the thermophilic Bacillus PS3 affects catalytic site cooperativity by destabilizing the closed conformation of the catalytic site

Fluorescence titrations of the alpha(3)(betaG(156)C/Y(345)W)(3)gamma, alpha(3)(betaE(199)V/Y(345)W)(3)gamma, and alpha(3)(betaY(345)W)(3)gamma subcomplexes of TF(1) with nucleotides show that the betaG(156)C substitution substantially lowers the affinity of catalytic sites for ATP and ADP with or without Mg(2+), whereas the betaE(199)V substitution increases the affinity of catalytic sites for nucleotides. Whereas the alpha(3)(betaG(156)C)(3)gamma and alpha(3)(betaE(199)V)(3)gamma subcomplexes hydrolyze 2 mM ATP at 2% and 0.7%, respectively, of the rate exhibited by the wild-type enzyme, the alpha(3)(betaG(156)C/E(199)V)(3)gamma hydrolyzes 2 mM ATP at 9% the rate exhibited by the wild-type enzyme. The alpha(3)(betaG(156)C)(3)gamma, alpha(3)(betaG(156)C/E(199)V)(3)gamma, and alpha(3)(betaG(156)C/E(199)V/Y(345)W)(3)gamma subcomplexes resist entrapment of inhibitory MgADP in a catalytic site during turnover. Product [(3)H]ADP remains tightly bound to a single catalytic site when the wild-type, betaE(199)V, betaY(345)W, and betaE(199)V/Y(345)W subcomplexes hydrolyze substoichiometric [(3)H]ATP, whereas it is not retained by the betaG(156)C and betaG(156)C/Y(345)W subcomplexes. Less firmly bound, product [(3)H]ADP is retained when the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze substoichiometric [(3)H]ATP. The Lineweaver-Burk plot obtained with the betaG(156)C mutant is curved downward in a manner indicating that its catalytic sites act independently during ATP hydrolysis. In contrast, the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze ATP with linear Lineweaver-Burk plots, indicating cooperative trisite catalysis. It appears that the betaG(156)C substitution destabilizes the closed conformation of a catalytic site hydrolyzing MgATP in a manner that allows release of products in the absence of catalytic site cooperativity. Insertion of the betaE(199)V substitution into the betaG(156)C mutant restores cooperativity by restricting opening of the catalytic site hydrolyzing MgATP for product release until an open catalytic site binds MgATP.     

Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F1 ATPase (CF1) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. We have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg2+ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF1 that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF1. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (Pi) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with [32P]Pi, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. We also report the occurrence of a 1-2-min delay in the onset of the Mg2+-induced inhibition after addition of CF1 to solutions containing Mg2+ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of Pi formation is followed by a much lower, constant steady-state rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase

The crystal structure of yeast mitochondrial F(1) ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the alpha(3)beta(3) sub-assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F(1) ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F(1) ATPase has an ATP analog bound and therefore this structure does not represent the ADP-inhibited form. In addition, one of the complexes binds phosphate in the nucleotide-free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F(1) ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis.     

Reaction mechanism of the membrane-bound ATPase of submitochondrial particles from beef heart

Submitochondrial particles from beef heart, washed with dilute solutions of KCl so as to activate the latent, membrane-bound ATPase, F1, may be used to study single site catalysis by the enzyme. [gamma-32P]ATP, incubated with a molar excess of catalytic sites, a condition which favors binding of substrate in only a single catalytic site on the enzyme, is hydrolyzed via a four-step reaction mechanism. The mechanism includes binding in a high affinity catalytic site, Ka = 10(12)M-1, a hydrolytic step for which the equilibrium constant is near unity, and two product release steps in which Pi dissociates from catalytic sites about 10 times more rapidly than ADP. Catalysis by the membrane-bound ATPase also is characterized by a 10(6)-fold acceleration in the rate of net hydrolysis of [gamma-32P]ATP, bound in the high affinity catalytic site, that occurs when substrate is made available to additional catalytic sites on the enzyme. These aspects of the reaction mechanism of the ATPase of submitochondrial particles closely parallel the reaction mechanism determined for solubilized, homogeneous F1 (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100). The finding that removal of the enzyme from the membrane does not significantly alter the properties of single site catalysis lends support to models of ATP synthesis in oxidative phosphorylation, catalyzed by membrane-bound F1, that have been based on the study of the soluble enzyme.     

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.     

The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase.

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.     

Characteristics of the combination of inhibitory Mg2+ and azide with the F1 ATPase from chloroplasts.

The interactions between ADP, Mg2+, and azide that result in the inhibition of the chloroplast F1 ATPase (CF1) have been explored further. The binding of the inhibitory Mg2+ with low Kd is shown to occur only when tightly bound ADP is present at a catalytic site. Either the tightly bound ADP forms part of the Mg(2+)-binding site or it induces conformational changes creating the high-affinity site for inhibitory Mg2+. Kinetic studies show that CF1 forms two catalytically inactive complexes with Mg2+. The first complex results from Mg2+ binding with a Kd for Mg2+ dissociation of about 10-15 microM, followed by a slow conversion to a complex with a Kd of about 4 microM. The rate-limiting step of the CF1 inactivation by Mg2+ is the initial Mg2+ binding. When medium Mg2+ is chelated with EDTA, the two complexes dissociate with half-times of about 1 and 7 min, respectively. Azide enhances the extent of Mg(2+)-dependent inactivation by increasing the affinity of the enzyme for Mg2+ 3-4 times and prevents the reactivation of both complexes of CF1 with ADP and Mg2+. This results from decreasing the rate of Mg2+ release; neither the rate of Mg2+ binding to CF1 nor the rate of isomerization of the first inactive complex to the more stable form is affected by azide. This suggests that the tight-binding site for the inhibitory azide requires prior binding of both ADP and Mg2+.