Exchange and hydrolysis of tightly bound nucleotides in normal and photolabelled bovine heart mitochondrial F1-ATPase

Treatment of F1 by threefold fast-column centrifugation or by single ammonium sulphate precipitation followed by fast-column centrifugation resulted in enzyme preparations containing 2.5-2.8 mol of bound nucleotides per mol of F1. Short incubations of such F1 preparations in the presence of relatively low concentrations of [14C]ATP and 2-azido[alpha-32P]ATP (100-250 microM), followed by ammonium sulphate precipitation and fast-column centrifugation, resulted in exchange of about 1 mol of the bound nucleotide per mol of F1 not affecting the total amount of bound nucleotides. Exchange of bound nucleotides with 2-azidoATP, followed by ultraviolet irradiation, results in inhibition of the enzyme activity, full inhibition being obtained (via extrapolation) when 1 mol of 2-nitreno-adenosine 5'-tri- or diphosphate (2-N-AT(D)P) is covalently bound to the presumably catalytic site on the enzyme (Van Dongen, M.B.M., De Geus, J.P., Korver, T., Harton, A.F. and Berden, J.A. (1986) Biochim. Biophys. Acta 850, 359-368). In agreement with this, it was found that incorporated [gamma-32P]ATP was hydrolysed by more than 80%. Newly incorporated, not covalently bound radioactive nucleotides could be rapidly exchanged again by the addition of non-radioactive nucleotides, but a higher concentration of nucleotides was needed to fully exchange the incorporated nucleotide. Also, when F1 was depleted of most of its bound nucleotides by repeated ammonium sulphate precipitation, part of the residual nucleotides was still rapidly exchangeable. The ability of F1 to exchange (and hydrolyse) one of the bound nucleotides was not lost when one catalytic and one non-catalytic binding site were occupied by covalently bound 8-N-ATP. Similar results were obtained with F1 containing 2-nitrenoATP covalently bound to one of the catalytic sites. Also, after photolabelling of up to four binding sites with 8-N[( 2-3H]AT(D)P, part of the two remaining non-covalently bound nucleotides could still be rapidly exchanged. In this case the exchanged nucleotide was also hydrolysed. It is concluded that one of the two bound nucleotides became exchangeable when all four other sites (i.e., two catalytic and two non-catalytic) were occupied with covalently bound nucleotides. The site involved showed catalytic properties suggestive of localisation on a beta-subunit.     

Structural changes in mitochondrial F1-ATPase induced by the removal of loosely bound nucleotides

Mitochondrial F1-ATPase from beef heart, forms aggregates when it is depleted of loosely bound nucleotides by repeated precipitation in ammonium sulfate. Polyacrylamide gradient gel electrophoresis, in non dissociating conditions shows that the aggregate formed is a dimer (708,000 daltons). The aggregation is attributed to a conformational change of the protein as a consequence of the elimination of the nucleotides from the low affinity binding sites. This structural alteration seems to be reversible because, after addition of ATP, the aggregation is not observed on polyacrylamide gels but the catalytic properties remain unchanged. This conformational change alters the accessibility of protein sulfhydryl groups to 5,5' - dithiobis(2-nitrobenzoic acid). All these observations emphasize the importance of protein nucleotide interactions to the conformation of the mitochondrial F1-ATPase.     

Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.     

Excessive ATP hydrolysis in ischemic myocardium by mitochondrial F1F0-ATPase: effect of selective pharmacological inhibition of mitochondrial ATPase hydrolase activity

Mitochondrial F(1)F(0)-ATPase normally synthesizes ATP in the heart, but under ischemic conditions this enzyme paradoxically causes ATP hydrolysis. Nonselective inhibitors of this enzyme (aurovertin, oligomycin) inhibit ATP synthesis in normal tissue but also inhibit ATP hydrolysis in ischemic myocardium. We characterized the profile of aurovertin and oligomycin in ischemic and nonischemic rat myocardium and compared this with the profile of BMS-199264, which only inhibits F(1)F(0)-ATP hydrolase activity. In isolated rat hearts, aurovertin (1-10 microM) and oligomycin (10 microM), at concentrations inhibiting ATPase activity, reduced ATP concentration and contractile function in the nonischemic heart but significantly reduced the rate of ATP depletion during ischemia. They also inhibited recovery of reperfusion ATP and contractile function, consistent with nonselective F(1)F(0)-ATPase inhibitory activity, which suggests that upon reperfusion, the hydrolase activity switches back to ATP synthesis. BMS-199264 inhibits F(1)F(0) hydrolase activity in submitochondrial particles with no effect on ATP synthase activity. BMS-199264 (1-10 microM) conserved ATP in rat hearts during ischemia while having no effect on preischemic contractile function or ATP concentration. Reperfusion ATP levels were replenished faster and necrosis was reduced by BMS-199264. ATP hydrolase activity ex vivo was selectively inhibited by BMS-199264. Therefore, excessive ATP hydrolysis by F(1)F(0)-ATPase contributes to the decline in cardiac energy reserve during ischemia and selective inhibition of ATP hydrolase activity can protect ischemic myocardium.     

The binding of a second divalent metal ion is necessary for the activation of ATP hydrolysis and its inhibition by tightly bound ADP in the ATPase from Halobacterium saccharovorum.

A binding site for divalent metal ions on the ATPase from Halobacterium saccharovorum is proposed. This site is different from the catalytic site which binds ATP and a complexed divalent metal ion. Occupation of the second site greatly stimulates the rate of ATP hydrolysis and the affinity of the catalytic site for the metal ion-ATP complex. The time-dependent inhibition of the ATPase, which occurs during catalysis and which is known to be caused by the retention of ADP, is also dependent on the occupation of this metal ion binding site. The binding of the metal ion apparently induces extremely tight binding of ADP after the departure of Pi. Mg2+, Mn2+, Zn2+, Co2+, and Ca2+ were tested and showed both the activating and the inhibitory effects, although their binding constants for ATP and the second metal ion binding site were quite different. The characteristic shapes of the nonlinear ATP hydrolysis curves obtained with different metal ions, and different ratios of metal ion and ATP, could be explained with the established dissociation constants. On this basis, a model for the ATPase was developed with two catalytic cycles: one in which the second metal ion binding site is occupied, and another in which it is empty. These pathways are connected by metal ion-dependent equilibria.     

A structure-based model for the synthesis and hydrolysis of ATP by F1-ATPase

Many essential functions of living cells are performed by nanoscale protein motors. The best characterized of these is F(o)F1-ATP synthase, the smallest rotary motor. This rotary motor catalyzes the synthesis of ATP with high efficiency under conditions where the reactants (ADP, H2PO4(-)) and the product (ATP) are present in the cell at similar concentrations. We present a detailed structure-based kinetic model for the mechanism of action of F1-ATPase and demonstrate the role of different protein conformations for substrate binding during ATP synthesis and ATP hydrolysis. The model shows that the pathway for ATP hydrolysis is not simply the pathway for ATP synthesis in reverse. The findings of the model also explain why the cellular concentration of ATP does not inhibit ATP synthesis.     

Viscosimetric analysis of the mechanism of ATP hydrolysis by pea chloroplast F1-ATPase

Photosynthetica - Dependence of ATP hydrolysis kinetics by the chloroplast coupling factor (CF1) on medium viscosity was studied at varying temperatures. For samples with oxidized and reduced CF1...     

Uni-site catalysis by Escherichia coli F1-ATPase with different numbers of bound nucleotides

We prepared two types of E. coli F1 by slightly different gel filtration procedures of the purified F1: F1(II) contained about 2 mol, and F1(V) about 5 mol of bound adenine nucleotides per mol of the enzyme. Thus F1(II) had more than 2, possibly 3, vacant catalytic sites, while F1(V) had less than one vacant catalytic site. The rate of ATP hydrolysis in uni-site catalysis (in the presence of inorganic phosphate) was about 3-fold higher with F1(II) than with F1(V), suggesting that ADP and inorganic phosphate bound at the catalytic sites of F1(V) changed the kinetics of uni-site catalysis significantly.     

Heterogeneous hydrolysis of substoichiometric ATP by the F1-ATPase from Escherichia coli.

The hydrolysis of 0.3 microM [alpha,gamma-32P]ATP by 1 microM F1-ATPase isolated from the plasma membranes of Escherichia coli has been examined in the presence and absence of inorganic phosphate. The rate of binding of substoichiometric substrate to the ATPase is attenuated by 2 mM phosphate and further attenuated by 50 mM phosphate. Under all conditions examined, only 10-20% of the [alpha,gamma-32P]ATP that bound to the enzyme was hydrolyzed sufficiently slowly to be examined in cold chase experiments with physiological concentrations of non-radioactive ATP. These features differ from those observed with the mitochondrial F1-ATPase. The amount of bound substrate in equilibrium with bound products observed in the slow phase which was subject to promoted hydrolysis by excess ATP was not affected by the presence of phosphate. Comparison of the fluxes of enzyme-bound species detected experimentally in the presence of 2 mM phosphate with those predicted by computer simulation of published rate constants determined for uni-site catalysis (Al-Shawi, M.D., Parsonage, D. and Senior, A.E. (1989) J. Biol. Chem. 264, 15376-15383) showed that hydrolysis of substoichiometric ATP observed experimentally was clearly biphasic. Less than 20% of the substoichiometric ATP added to the enzyme was hydrolyzed according to the published rate constants which were calculated from the slow phase of product release in the presence of 1 mM phosphate. The majority of the substoichiometric ATP added to the enzyme was hydrolyzed with product release that was too rapid to be detected by the methods employed in this study, indicating again that the F1-ATPase from E. coli and bovine heart mitochondria hydrolyze substoichiometric ATP differently.     

ATP hydrolysis in the betaTP and betaDP catalytic sites of F1-ATPase

The enzyme F1-adenosine triphosphatase (ATPase) is a molecular motor that converts the chemical energy stored in the molecule adenosine triphosphate (ATP) into mechanical rotation of its gamma-subunit. During steady-state catalysis, the three catalytic sites of F1 operate in a cooperative fashion such that at every instant each site is in a different conformation corresponding to a different stage along the catalytic cycle. Notwithstanding a large amount of biochemical and, recently, structural data, we still lack an understanding of how ATP hydrolysis in F1 is coupled to mechanical motion and how the catalytic sites achieve cooperativity during rotatory catalysis. In this publication, we report combined quantum mechanical/molecular mechanical simulations of ATP hydrolysis in the betaTP and betaDP catalytic sites of F1-ATPase. Our simulations reveal a dramatic change in the reaction energetics from strongly endothermic in betaTP to approximately equienergetic in betaDP. The simulations identify the responsible protein residues, the arginine finger alphaR373 being the most important one. Similar to our earlier study of betaTP, we find a multicenter proton relay mechanism to be the energetically most favorable hydrolysis pathway. The results elucidate how cooperativity between catalytic sites might be achieved by this remarkable molecular motor.     

Steady-state rate of F1-ATPase turnover during ATP hydrolysis by the single catalytic site

Under the conditions of ATP regeneration and molar excess of nucleotide-depleted F1-ATPase the enzyme catalyses steady-state ATP hydrolysis by the single catalytic site. Values of Km = 10(-8) M and Vm = 0.05 s-1 for the single-site catalysis have been determined. ADP release limits single-site ATP hydrolysis under steady-state conditions. The equilibrium constant for ATP hydrolysis at the F1-ATPase catalytic site is less than or equal to 0.7.     

Steady-state kinetics of F1-ATPase. Mechanism of anion activation

The kinetic behaviour of the ATPase activity of beef heart F1 depends largely on the exposure of the enzyme to some anionic ligands such as sulphate and/or EDTA. F1 prepared in the presence of such anions exhibited a triphasic kinetic pattern whereas F1 from which those anions were removed by dialysis exhibited only two Km values for ATP. Conversely to what has been previously reported, bicarbonate did not linearize F1-ATPase kinetics. Moreover, anion activation cannot be simply explained by promotion of ADP release but mainly by an increase in affinity of the third catalytic site for ATP.     

Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis

The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abolition of anion-activation of mitochondrial F1-ATPase by the partial ADP-induced hysteretic inhibition

An M_r 21 000 polypeptide, designated APPG, has been purified by reverse-phase, high-performance liquid chromatography (RP-HPLC), from acid extracts of porcine anterior pituitary glands. This acidic protein possesses an isoelectric point of 4.9. Amino acid analysis shows that it is not a glycoprotein and estimates it to contain about 173 amino acids. NH₂-terminal sequence analysis allowed the determination of the first 50 residues unambiguously. A computer data bank search using a mutation data matrix and comparison with 269 012 protein segments indicated that this is a novel polypeptide sequence. However, this search revealed suggestive sequence homologies to a number of peptides of known sequence, including duck proinsulin (30%), Rous sarcoma virus transforming protein TVFV60 (24%) and pig secretin (26%).     

Evidence for catalytic cooperativity during ATP hydrolysis by beef heart F1-ATPase. Kinetics and binding studies with the photoaffinity label BzATP

The photoaffinity analog of ATP, 3'-O-(4-benzoyl) benzoyl ATP (BzATP), was used to covalently modify the catalytic sites on the beef heart mitochondrial F1-ATPase. In the absence of actinic illumination, BzATP was a slow substrate for the enzyme (Vmax = 0.19 mumol min-1 mg-1; kcat/Km = 2.2 X 10(6) M-1s-1) and behaved as a classical competitive inhibitor versus ATP (Ki = 0.85 microM). Under photolytic conditions, BzATP inactivated F1 with pseudo first-order kinetics, and the photoinactivation reaction showed rate saturation suggesting specific, reversible binding of BzATP to F1 prior to covalent bond formation. ATP protected against F1 photoinactivation (Kprotect = 0.3 microM) and partially covalently modified F1 yielded the same Km for ATP as unmodified enzyme. These results strongly suggested that BzATP was bound to catalytic sites on the enzyme. In the absence of photolysis, BzATP saturated two binding sites on the F1 (KD = 1.6 microM), and under photolytic conditions, 1 mol of BzATP was shown to be covalently liganded to the beta subunit of the enzyme coincident with 100% loss in ATPase activity. Previous studies with the mitochondrial F1-ATPase have suggested a mechanism involving catalytic cooperativity during ATP hydrolysis. Our demonstration of a molar stoichiometry of 1 for photoinactivation is in accord with this mechanism. It is suggested that either F1 is unable to hydrolyze covalently bound BzATP, or that subsequent to hydrolysis, the BzADP product can not be released from the catalytic site. It is therefore inferred that F1 hydrolytic activity requires cooperativity between multiple, viable catalytic sites and that covalent modification of a single catalytic site is sufficient for complete enzyme inactivation.     

Complex cooperativity of ATP hydrolysis in the F(1)-ATPase molecular motor

F(1)-ATPase catalyses ATP hydrolysis and converts the cellular chemical energy into mechanical rotation. The hydrolysis reaction in F(1)-ATPase does not follow the widely believed Michaelis-Menten mechanism. Instead, the hydrolysis mechanism behaves in an ATP-dependent manner. We develop a model for enzyme kinetics and hydrolysis cooperativity of F(1)-ATPase which involves the binding-state changes to the coupling catalytic reactions. The quantitative analysis and modeling suggest the existence of complex cooperative hydrolysis between three different catalysis sites of F(1)-ATPase. This complexity may be taken into account to resolve the arguments on the binding change mechanism in F(1)-ATPase.     

Removal of "tightly bound" nucleotides from soluble mitochondrial adenosine triphosphatase (F1).

Soluble mitochondrial ATPase (F1) from beef heart prepared in this laboratory contained approximately 1.8 mol of ADP and 0 mol of ATP/mol of F1 which were not removed by repeated precipitation of the enzyme with ammonium sulfate solution or by gel filtration in low ionic strength buffer containing EDTA. This enzyme had full coupling activity. Treatment of the enzyme with trypsin (5 mug/mg of F1 for 3 min) reduced the "tightly bound" ADP to zero, abolished coupling activity, but had no effect on the ATPase activity, stability, or membrane-binding capability of the F1. When the trypsin concentration was varied between 0 and 5 mug/mg of F1, tightly bound ADP was removed to varying degrees, and a correlation was seen between amount of residual tightly bound ADP and residual coupling activity. Gel filtration of the native F1 in high ionic strength buffer containing EDTA also caused complete loss of tightly bound ADP and coupling ability, whereas ATPase activity, stability, and membrane-binding capability were retained. The ADP-depleted F1 preparations were unable to rebind normal amounts of ADP or any ATP in simple reloading experiments. The results strongly suggest that tightly bound ADP is required for ATP synthesis and for energy-coupled ATP hydrolysis on F1. The results also suggest that ATP synthesis and energy-linked ATP hydrolysis rather than involving one nucleotide binding site on F1, involve a series or "cluster" of sites. The ATP hydrolysis site may represent one component of this cluster. The results show that nonenergy-coupled ATP hydrolysis on F1 can occur in the absence of tightly bound ADP or ATP.     

Aurovertin fluorescence changes of the mitochondrial F1-ATPase during multi- and uni-site ATP hydrolysis

The aurovertin-F1 complex was used to monitor fluorescence changes of the mitochondrial adenosine triphosphatase during multi- and uni-site ATP hydrolysis. It is known that the fluorescence intensity of the complex is partially quenched by addition of ATP or Mg2+ and enhanced by ADP (Chang, T., and Penefsky, H. S. (1973) J. Biol. Chem. 248, 2746-2754). In the present study low concentrations of ATP (0.03 mM) induced a marked fluorescence quenching which was followed by a fast fluorescence recovery. This recovery could be prevented by EDTA or an ATP regenerating system. The rate of ATP hydrolysis by the aurovertin-F1 complex and the reversal of the ATP-induced fluorescence quenching were determined in these various conditions. ITP hydrolysis also resulted in fluorescence quenching that was followed by a recovery of fluorescence intensity. Under conditions for single site catalysis, fluorescence quenching was observed upon the addition of ATP. This strongly indicates that fluorescence changes in the aurovertin-F1 complex are due to the binding and hydrolysis of ATP at a catalytic site. Therefore the resulting ADP molecule bound at this catalytic site possibly induces the fluorescence recovery observed.