Adenine nucleotide binding sites on beef heart F1-ATPase. Asymmetry and subunit location

Previously we have shown that beef heart mitochondrial F1 contains a total of six adenine nucleotide binding sites. Three "catalytic" sites exchange bound ligand rapidly during hydrolysis of MgATP, whereas three "noncatalytic" sites do not. The noncatalytic sites behave asymmetrically in that a single site releases bound ligand upon precipitation of F1 with ammonium sulfate. In the present study, we find this same site to be the only noncatalytic site that undergoes rapid exchange of bound ligand when F1 is incubated in the presence of EDTA at pH 8.0. Following 1000 catalytic turnovers/F1, the site retains the unique capacity for EDTA-induced exchange, indicating that the asymmetric determinants are permanent and that the three noncatalytic sites on soluble F1 do not pass through equivalent states during catalysis. Measurements of the rate of ligand binding at the unique noncatalytic site show that uncomplexed nucleotide binds preferentially. At pH 7.5, in the presence of Mg2+, the rate constant for ADP binding is 9 X 10(3) M-1 s-1 and for dissociation is 4 X 10(-4) s-1 to give a Kd = 50 nM. The rate of dissociation is 10 times faster in the presence of EDTA or during MgATP hydrolysis, and it increases rapidly at pH below 7. EDTA-induced exchange is inhibited by Mg2+, Mn2+, Co2+, and Zn2+ but not by Ca2+ and is unaffected by dicyclohexylcarbodiimide modification. The unique noncatalytic site binds 2-azido-ADP. Photolysis results in the labeling of the beta subunit. Photolabeling of a single high-affinity catalytic site under conditions for uni-site catalysis also results in the labeling of beta, but a different pattern of labeled peptides is obtained in proteolytic digests. The results demonstrate the presence of two different nucleotide binding domains on the beta subunit of mitochondrial F1.     

Adenine nucleotide-binding sites on beef heart F1-ATPase. Conditions that affect occupancy of catalytic and noncatalytic sites

Beef heart mitochondrial F1 contains a total of six adenine nucleotide-binding sites including at least two different types of sites. Three "exchangeable" sites exchange rapidly during hydrolysis of MgATP, whereas three "nonexchangeable" sites do not (Cross, R. L. and Nalin, C. M. (1982) J. Biol. Chem. 257, 2874-2881). When F1 that has been stored as a suspension in (NH4)2SO4/ATP/EDTA/sucrose/Tris, pH 8.0, is pelleted, rinsed with (NH4)2SO4, dissolved, and desalted, it retains three bound adenine nucleotides. We find that two of these endogenous nucleotides are bound at nonexchangeable sites and one at an exchangeable site. The vacant nonexchangeable site is highly specific for adenine nucleotide and is rapidly filled by ADP upon addition of ADP or during ATP hydrolysis. ADP bound at this site can be removed by reprecipitating the enzyme with (NH4)2SO4. The single nucleotide retained by desalted F1 at an exchangeable site is displaced during hydrolysis of ATP, GTP, or ITP. The binding of PPi at two sites on the enzyme also promotes its dissociation. Neither procedure affects retention of nucleotide at the nonexchangeable sites. These observations, combined with the finding that PPi is much more easily removed from exchangeable sites than ADP, have led to the development of a procedure for preparing F1 with uniform and well-defined nucleotide site occupancy. This involves sequential exposure to MgATP, PPi, and high concentrations of Pi. Unbound ligand is removed between each step. The resulting enzyme, F1[3,0], has three occupied nonexchangeable sites and three vacant exchangeable sites. Evidence that nonexchangeable and exchangeable sites represent noncatalytic and catalytic sites, respectively, suggest that this form of the enzyme will prove useful in numerous applications, including transient kinetic measurements and affinity labeling of active site residues.     

Effect of the epsilon-subunit on nucleotide binding to Escherichia coli F1-ATPase catalytic sites.

The influence of the epsilon-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (beta-Trp-331). The interaction between epsilon and F1 was not affected by the mutation. Kd for binding of epsilon to betaY331W mutant F1 was approximately 1 nM, and epsilon inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the epsilon-depleted and epsilon-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. Kd1(MgATP) and Kd1(MgADP) were an order of magnitude higher in the absence of epsilon than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the epsilon-depleted and epsilon-replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of epsilon, Km equals Kd3. Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (Vmax) MgATP hydrolysis rates.     

1H-NMR studies on nucleotide binding to the catalytic sites of bovine mitochondrial F1-ATPase

The conformation of adenine nucleotides bound to bovine mitochondrial F1-ATPase was investigated using transfer nuclear Overhauser enhancement measurements. It is shown that all nucleotides investigated adopt a predominantly anti conformation when bound to the catalytic sites. Furthermore, the experiment suggests that 8-azido-ADP and 8-azido-ATP, which are predominantly in the syn conformation in solution, are in the anti conformation when bound to F1 catalytic sites.     

Classification of nucleotide binding sites on mitochondrial F1-ATPase from yeast

Methods are described to classify nucleotide binding sites of the mitochondrial coupling factor F1 from yeast on the basis of their affinities and stability properties. High affinity sites or states for ATP and related adenine analogs and low affinity sites or states which bind a broad range of different nucleotide triphosphates are found. The results are discussed in terms of a two site, two cycle scheme, where binding of nucleotide at one site facilitates the release of nucleotide at a second site.     

Adenine nucleotide-binding sites on mitochondrial F1-ATPase. Evidence for an adenylate kinase-like orientation of catalytic and noncatalytic sites.

Nucleotide-depleted mitochondrial F1-ATPase (F1[0,0]) is inhibited by the diadenosine oligophosphate compounds, AP4A, AP5A, and AP6A (where APxA stands for 5',5'-diadenosine oligophosphates having a chain of x phosphoryl groups linking the two adenosine moieties). When F1[0,0] is preincubated with these compounds and then assayed for ATP hydrolysis activity under conditions that normally allow turnover at all three catalytic sites, the maximal level of inhibition observed is 80%. However, when assayed at lower ATP concentrations under conditions that allow simultaneous turnover at only two of the three sites, no inhibition is observed. A decrease in the number of phosphoryl groups that links the adenosine moieties to less than 4 (AP3A, AP2A) converts the compound to an activator of ATP hydrolysis, similar in effect to that obtained when one mol of ADP or 2-azido-ADP binds at a catalytic site on F1[0,0]. Inhibition by the compounds requires the presence of at least one vacant noncatalytic site. Evidence is provided that the probes also interact with a catalytic site. The stoichiometry for maximal inhibition by AP4A is 0.94 mol/mol of F1. The data presented support a model for the structure of nucleotide-binding sites on F1 that places catalytic and noncatalytic sites in close proximity in an orientation analogous to the ATP and AMP binding sites on adenylate kinase. Inhibition of the enzyme by the dinucleotide compounds can be explained by the cross-bridging of one of the catalytic sites to a noncatalytic site in analogy to the inhibition of adenylate kinase by AP5A. The residual capacity for bi-site catalysis indicates that the second and third catalytic sites remain catalytically active.     

Adenine nucleotide-binding sites on mitochondrial F1-ATPase: studies of the inactive complex formed upon binding ADP at a catalytic site.

ADP-induced inhibition of mitochondrial F1-ATPase has been studied. It is shown that in the presence of magnesium and the absence of light, the photoaffinity ADP analog, 2-azido-ADP, induces a reversible inhibition of native F1 that is indistinguishable from that obtained with ADP. Photolysis of the inactive complex results in the predominant labeling of a catalytic-site peptide identified previously (Cross et al., 1987, Proc. Natl. Acad. Sci. USA 84, 5715-5719). Dissociation of the inactive complex formed between F1 and ADP is biphasic with a rapid azide-insensitive phase followed by a slow azide-sensitive phase (k approximately 3 x 10(-3) s-1). It is also shown that incubation of the ADP-inhibited enzyme with EDTA or phosphate does not result in release or migration of ADP from the catalytic site. However, it does convert the complex to a form that reactivates in the presence of 100 microM ATP at a rate too rapid to observe using manual mixing.     

Site-site interaction on mitochondrial F1-ATPase. Functional symmetry of the high-affinity nucleotide binding sites

Interactions between the high affinity binding sites on mitochondrial F1 were analysed by combined use of the nucleotide analogues 3'-O-(1-naphthoyl)-ADP (N-ADP) and 2'-3'-O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP). The binding behaviour of F1 with respect to these ligands was studied by measuring the fluorescence of F1 and of TNP-ADP and the fluorescence anisotropy of N-ADP. A total of 3 high affinity binding sites can be occupied by TNP-ADP. By exchange experiments, it could be shown that binding of TNP-ADP to such a site considerably accelerates the dissociation of a ligand bound to a neighbouring site. These results support the notion that the functional behaviour of F1 is symmetric: during the catalytic cycle any individual site can successively assume different affinity states as has been predicted by hypotheses such as the binding change model.     

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.     

Evidence for a nucleotide binding site on the isolated beta subunit from Escherichia coli F1-ATPase. Interaction between nucleotide and aurovertin D binding sites

The nucleotide binding capacity and affinity of the isolated beta subunit from Escherichia coli F1-ATPase have been studied with radiolabeled ADP and ATP by an equilibrium dialysis technique. Each mole of beta subunit in the presence of EDTA bound 1 mol of ADP or ATP with Kd values of 25 microM and 50-100 microM, respectively. At a saturating concentration, aurovertin enhanced the affinity of ADP or ATP for the isolated beta subunit by 3-6-fold. The Kd values for the binding of ADP or ATP were also assessed through the enhancing effect of ADP on [14C]aurovertin binding (Issartel, J.-P., Klein, G., Satre, M., & Vignais, P.V. (1983) Biochemistry 22, 3485-3492); the Kd values determined by this approach were several times lower than in the absence of aurovertin, in agreement with results obtained by direct titration with radiolabeled ADP or ATP.     

Exploration of delta-subunit interactions in beef heart mitochondrial F1-ATPase by monoclonal antibodies

Three monoclonal antibodies (mAbs) recognizing distinct epitopes on the delta-subunit of beef heart mitochondrial F1-ATPase were studied for their reactivity towards the delta-subunit both in isolated F1 and in the F0-F1 complex of submitochondrial particles. Two of the antibodies termed mAb delta 195 and mAb delta 239 had free access to delta in F1 and the F0-F1 complex. Partial hindrance was observed for the third antibody mAb delta 22. By a double antibinding assay, it was found that the binding sites for mAb delta 195 and mAb delta 239 were close to each other and possibly overlapping. Mapping studies conducted with the isolated delta-subunit showed that mAb delta 195 and mAb delta 239 interacted with the N-terminal portion of delta extending from Ala-1 to Met-16, whereas mAb delta 22 interacted with the fragment spanning Ser-17-Glu-68. It was concluded that the Ala-1-Met-16 segment of the delta-subunit in F1 and the F0-F1 complex is freely accessible from the outside, whereas the Ser-17-Glu-68 segment of delta is partially hidden, possibly as a result of interactions with other subunits.     

Further investigations on the inorganic phosphate binding site of beef heart mitochondrial F1-ATPase

The possibility that 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (Pi) [Lauquin, G., Pougeois, R., & Vignais, P. V. (1980) Biochemistry 19, 4620-4626], could mimic ATP was investigated. ANPP was hydrolyzed in the dark by sarcoplasmic reticulum Ca2+-ATPase in the presence of Ca2+ but not in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANPP was not hydrolyzed by purified mitochondrial F1-ATPase; however, ADP and ATP protected F1-ATPase against ANPP photoinactivation. On the other hand, the trinitrophenyl nucleotide analogues (TNP-ADP, TNP-ATP, and TNP-AMP-PNP), which bind specifically at the two catalytic sites of F1-ATPase [Grubmeyer, C., & Penefsky, H. (1981) J. Biol. Chem. 256, 3718-3727], abolished Pi binding on F1-ATPase; they do not protect F1-ATPase against ANPP photoinactivation. Furthermore, ANPP-photoinactivated F1-ATPase binds the TNP analogues in the same way as the native enzyme. The Pi binding site of F1-ATPase, which is shown to be photolabeled by ANPP, does not appear to be at the gamma-phosphate position of the catalytic sites.     

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.     

Electron microscopy of beef heart mitochondrial F1-ATPase

The quaternary structure of isolated and membrane-bound F1-ATPase (submitochondrial particles) has been studied by electron microscopy. A model of the molecule has been proposed: six protein masses are arranged in two layers approximately at the vertices of a triangular antiprism. Computer averaging of the images showed that the frontal view of the molecule can be approximately characterized by mirror plane symmetry.     

Electrostatic interactions in catalytic centers of F1-ATPase

The first part of this paper is a brief review of works concerned with the mechanisms of functioning of F0F1-ATP synthases. F0F1-ATP syntheses operate as rotating molecular machines that provide the synthesis of ATP from ADP and inorganic phosphate (Pi) in mitochondria, chloroplasts, and bacteria at the expense of the energy of electrochemical gradient of hydrogen ions generated across energy-transducing mitochondrial, chloroplast or, bacterial membranes. A distinguishing feature of these enzymes is that they operate as rotary molecular motors. In the second part of the work, we calculated the contribution of electrostatic interactions between charged groups of a substrate (MgATP), reaction products (MgADP and Pi), and charged amino acid residues of the F1-ATPase molecule to energy changes associated with the binding of ATP and its chemical transformations in the catalytic centers located at the interface of the alpha- and beta-subunits of the enzyme (oligomer complex alpha 3 beta 3 gamma of bovine mitochondrial ATPase). The catalytic cycle of ATP hydrolysis considered in the work includes conformational changes of alpha- and beta-subunits caused by unidirectional rotations of the central gamma-subunit. The results of our calculations are consistent with the idea that the energetically favorable process of ATP binding to the "open" catalytic center of F1-ATPase initiates the rotation of the gamma-subunit followed by ATP hydrolysis in another ("closed") catalytic center of the enzyme.     

Kinetic studies on rat liver and beef heart mitochondrial ATPase. Evidence for nucleotide binding at separate regulatory and catalytic sites.

Mitochondrial ATPases from rat liver and beef heart were used to study the effects of guanylylimidodiphosphate (GMP-P(NH)P) and adenylylimidodiphosphate (AMP-P(NH)P) on the kinetics of MgATP, MgITP, and MgGTP hydrolysis. AMP-P(NH)P was a noncompetitive inhibitor of hydrolysis of all substrates with the rat liver enzyme, whether activating anions were present or not. Also with the liver enzyme, AMP-P(NH)P caused only MgATP hydrolysis to appear to have positive cooperativity. With the beef heart enzyme, AMP-P(NH)P was a competitive inhibitor of ATPase activity and caused positive cooperativity; it gave noncompetitive patterns with GTP or ITP as substrates. In both enzyme systems, GMP-P(NH)P gave complex inhibition patterns with MgATP as the substrate, but was a competitive inhibitor of MgITP and MgGTP hydrolysis. These results are interpreted as indicating the existence of two types of nucleotide binding sites, with varying degrees of specificity and interaction on the ATPase molecules from both sources. It is postulated that MgATP and AMP-P(NH)P bind to regulatory site while MgATP, MgGTP, Mgitp, and GMP-P(NH)P bind to the catalytic site.