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1.
A kinetic analysis of ATP binding to noncatalytic sites of chloroplast coupling factor CF1 was made. The ATP binding proved to be unaffected by reduction of the disulfide bridge of the CF1 -subunit. The first-order equation describing nucleotide binding to noncatalytic sites allowed for two vacant nucleotide binding sites different in their kinetics. As suggested by nucleotide concentration dependence of the rate of nucleotide binding, the tight binding was preceded by rapid reversible binding of nucleotides. Preincubation of CF1 with Mg2+ resulted in a decreased rate of ATP binding. ATP dissociation from noncatalytic sites was described by the first order equation for similar sites with a dissociation rate constant k d (ATP) 10–3 min–1. Noncatalytic sites of CF1 were shown to be not homogeneous. One of them retained the major part of endogenous ADP after precipitation of CF1 with ammonium sulfate. Its two other sites differed in kinetic parameters and affinity for ATP. Anions of phosphate, sulfite, and especially, pyrophosphate inhibited the interaction between ATP and the noncatalytic sites.  相似文献   

2.
The interaction between sulfite, an efficient Mg2+-dependent F1-ATPase activator, and chloroplast CF1-ATPase was studied. The sulfite anion was shown to inhibit ADP and ATP binding to the noncatalytic sites of CF1. The stimulating activity of sulfite persists when all noncatalytic sites are nucleotide-occupied. Phosphate, a competing candidate for binding to CF1 catalytic sites, suppresses this activity. These results support the suggestion that the stimulation of Mg2+-dependent ATPase activity of CF1 is caused by sulfite binding to its catalytic sites.  相似文献   

3.
Two interesting previously reported properties of mitochondrial F1 ATPase have been confirmed and have been examined by18O exchange measurements to assess if they are consistent with sequential participation of catalytic sites during ATP hydrolysis. These are the ability of HCO 3 to increase reaction rate with apparent loss of cooperative interaction between subunits and the ability of ITP to accelerate the hydrolysis of a low concentration of ATP. The effect of HCO 3 was tested at concentrations of ATP lower than previous measurements. The activation disappeared when ATP was reduced to 0.1 µM. The HCO 3 activation at higher ATP concentrations did not change the extent of reversal of the cleavage of tightly bound ATP at the catalytic site, as measured by the average number of water oxygens incorporated with each Pi formed when 5 or 10 µM ATP is hydrolyzed. The data are consistent with sequential site participation with HCO 3 acceleration of ADP departure after a binding change that stops18O exchange and loosens ADP binding.When ITP concentration was lowered during net ITP hydrolysis by F1 ATPase an increase in water oxygen incorporation into Pi formed is observed, as noted previously for ATP hydrolysis. The acceleration of the cleavage of a constant low concentration of [-18O]ATP by concomitant hydrolysis of increasing concentrations of ITP was accompanied by a decrease in water oxygen incorporation with each Pi formed from the ATP. These results add to evidence for the binding change mechanism for F1 ATPase with sequential participation of catalytic sites.  相似文献   

4.
Incubation of the isolated H+-ATPase from chloroplasts, CF0F1, with 2-azido-[α-32P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (β-Tyr362) and non-catalytic (β-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 μM 2-azido-[α-32P]ATP, that with the intermediate dissociation constant (site 5) at 2 μM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[α-32P]ATP was possible. CF0F1 with non-covalently bound 2-azido-[α-32P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by ΔpH/Δϕ. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF0F1. Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between β- and α-subunits which is abolished by covalent derivatization of β-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F0-part and the F1-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three β-Tyr385.  相似文献   

5.
The pH-dependence of ADP and ATP affinity for CF1 tight nucleotide-binding sites was studied under conditions of equilibrium between bound and free labeled nucleotides. With the nucleotide/CF1 ratio>1, the ATP content in tightly bound nucleotides depended only slightly on medium pH. With the nucleotide/CF1 ratio approaching 1, tightly bound ATP content grew rapidly with decreasing pH. Calculations of ADP/ATP ratio in free and tightly bound nucleotides showed that decreasing the pH from 8.0 to 6.0 induced a 150 times greater affinity of the nucleotide-binding site for ATP than for ADP. The data indicates that ATP-ADP equilibrium at the CF1 tight nucleotide-binding site depends on protonation of specific acid-base groups of the enzyme.Abbreviations CF1, BF1, and MF1 coupling factors of chloroplasts, bacteria, and mitochondria, respectively - AdN adenine nucleotide  相似文献   

6.
《BBA》1985,809(1):27-38
8-Azido-ATP is a substrate for the ATP synthase in submitochondrial particles with a Vmax equal to 6% of the Vmax with ATP. The Km values for 8-azido-ATP are similar to those for ATP. ATP synthase in submitochondrial particles can bind maximally 2 mol 8-N-ATP or 8-N-ADP per mole and the inhibition of ATP hydrolysis by covalently bound N-ATP or N-ADP is proportional to the saturation of the enzyme with inhibitor, similar to the results obtained with isolated F1. Both 8-N-ATP and 8-N-ADP are bound mainly to the β subunits and at all levels of saturation the distribution of the label is 77% to the β and 23% to the α subunits. It is proposed that the binding of 8-azido-AXP itself is mainly to the β subunit, but that part of the nitreno radicals formed during excitation with light reacts with an amino acid of the α subunit, due to the location of the binding site at an interface between a β and an α subunit. Partial saturation with 8-N-ATP, under conditions that the concentration of 8-azido-ATP during the incubation is intermediate between the low and high Km values, does not abolish the apparent negative cooperativity of ATP hydrolysis. It is concluded that this apparent cooperativity is not due to the presence of two different catalytic sites, nor to a cooperativity between the two catalytic sites, but to interaction between the catalytic sites and regulatory sites.  相似文献   

7.
Nucleotide binding properties of two vacant noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1 (CF1) were studied. Kinetics of nucleotide binding to noncatalytic sites is described by the first-order equation that allows for two nucleotide binding sites that differ in kinetic features. Dependence of the nucleotide binding rate on nucleotide concentration suggests that tight nucleotide binding is preceded by rapid reversible binding of nucleotides. ADP binding is cooperative. The preincubation of CF1 with Mg2+ produces only slight effect on the rate of ADP binding and decreases the ATP binding rate. The ATP and ADP dissociation from noncatalytic sites is described by the first-order equation for similar sites with dissociation rate constants k−2(ADP)=1.5×10−1 min−1 and k−2(ATP)≅10−3 min−1, respectively. As follows from the study, the noncatalytic sites of CF1 are not homogeneous. One of them retains the major part of endogenous ADP after CF1 precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides.  相似文献   

8.
At concentrations below 1 mM, hydrophobic pyridine homologues decrease the rate of photophosphorylation and light-stimulated hydrolysis of ATP and light-activated exchange of the tightly bound nucleotides in chloroplasts, but increase the rate of the Hill reaction. Unlike uncoupling agents, the presence of the organic base at such low concentrations decreases the rate of light-dependent leakage and has no effect on the efficiency of two-stage photophosphorylation in broken chloroplasts. By assuming that the organic base is bound to independent equivalent sites in the thylakoid membrane, a simple expression can be derived which relates the observed rates of photophosphorylation and light-stimulated hydrolysis of ATP quantitatively to the concentration of the organic base in solution and gives dissociation equilibrium constants which are on the order of the relative hydrophobicities of the pyridine homologues. A possible mechanistic model for the CF0 · CF1 complex is proposed which could serve as the basis for a unified interpretation of the kinetics of proton translocation in illuminated chloroplasts, the steady-state rate of photophosphorylation, the light-stimulated ATPase activity, and the light-activated exchange of tightly bound adenine nucleotides.Abbreviations AMPPNP adenylylimidodiphosphate - Chl chlorophyll - CF0 · CF1 the coupling factor complex of chloroplasts - DCCD N,N-dicyclohexylcarbodiimide - DTT dithiothreitol - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - TCA trichloroacetic acid - Tricine N-tris-(hydroxymethyl)methylglycine  相似文献   

9.
A modified ‘cold chase’ technique was used to study tight [14C]ADP and [14C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF0F1). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF0F1 or CF1 reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k d of about 5 min−1. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF0F1. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF1 dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF1 becomes bound to a thylakoid membrane.  相似文献   

10.
ATP binding and hydrolysis are critical for protein degradation by HslUV, a AAA + machine containing one or two HslU6 ATPases and the HslV12 peptidase. Although each HslU homohexamer has six potential ATP-binding sites, we show that only three or four ATP molecules bind at saturation and present evidence for three functional subunit classes. These results imply that only a subset of HslU and HslUV crystal structures represents functional enzyme conformations. Our results support an asymmetric mechanism of ATP binding and hydrolysis, and suggest that molecular contacts between HslU and HslV vary dynamically throughout the ATPase cycle. Nucleotide binding controls HslUV assembly and activity. Binding of a single ATP allows HslU to bind HslV, whereas additional ATPs must bind HslU to support substrate recognition and to activate ATP hydrolysis, which powers substrate unfolding and translocation. Thus, a simple thermodynamic hierarchy ensures that substrates bind to functional HslUV complexes, that ATP hydrolysis is efficiently coupled to protein degradation, and that working HslUV does not dissociate, allowing highly processive degradation.  相似文献   

11.
《BBA》1987,893(2):275-288
The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, Eoxi. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, Ereda. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme Eredi is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, voxmax = 400 ATP per CF0F1 per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, vredmax = 400 ATP per CF0F1 per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis vredmax = −90 ATP per CF0F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase.  相似文献   

12.
《FEBS letters》1987,223(2):304-308
The ATP-hydrolyzing activity of F1-ATPase purified from potato tubers mitochondria was stimulated 2- and 3.5-fold by anions, chloride and bicarbonate, respectively, and 5.5- and 6.5-fold by detergents, octyl glucoside and lauryl dimethylamine oxide (LDAO), respectively. The maximal specific activity of the activated F1, 129 μmol/min per mg protein is the highest activity of plant mitochondrial F1 hitherto reported and exceeds several-fold values reported earlier. In the absence of activators F1-catalyzed ATP hydrolysis exhibits non-linear double-reciprocal plots of [ATP]−1 vs v−1 indicative of negative cooperativity, while in the presence of activators, linear plots are observed. It is suggested that the activators reduce the cooperativity originating from the interaction between different subunits of the enzyme.  相似文献   

13.
Vladimir V. Bulygin 《BBA》2009,1787(8):1016-1023
Nucleotide binding to nucleotide-depleted F1-ATPase from Escherichia coli (EcF1) during MgATP hydrolysis in the presence of excess ? subunit has been studied using a combination of centrifugal filtration and column-centrifugation methods. The results show that nucleotide-binding properties of catalytic sites on EcF1 are affected by the state of occupancy of noncatalytic sites. The ATP-concentration dependence of catalytic-site occupancy during MgATP hydrolysis demonstrates that a bi-site mechanism is responsible for the positive catalytic cooperativity observed during multi-site catalysis by EcF1. The results suggest that a bi-site mechanism is a general feature of F1 catalysis.  相似文献   

14.
Yakov M. Milgrom 《BBA》2010,1797(10):1768-1774
The effect of inorganic phosphate (Pi) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F1-ATPase from beef heart mitochondria (ndMF1) has been investigated. It is shown for the first time that Pi decreases the apparent rate constant of uni-site ATP binding by ndMF1 3-fold with the Kd of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, Pi also shifts equilibrium between bound ATP and ADP + Pi in the direction of ATP synthesis with the Kd of 0.17 ± 0.03 mM. However, 10 mM Pi does not significantly affect ATP binding during multi-site catalysis.  相似文献   

15.
We explored the concentration gradient effects of the sodium and lithium ions and the deuterium isotope's effects on the activities of H+-ATP synthase from chloroplasts (CF0F1). We found that the sodium concentration gradient can drive the ATP synthesis reaction of CF0F1. In contrast, the lithium ion can be an efficient enzyme-inhibitor by blocking the entrance channel of the ion translocation pathway in CF0. In the presence of sodium or lithium ions and with the application of a membrane potential, unexpected enzyme behaviors of CF0F1 were evident. To account for these observations, we propose that both of the sodium and lithium ions could undergo localized hydrolysis reactions in the chemical environment of the ion channel of CF0. The protons generated locally could proceed to complete the ion translocation process in the ATP synthesis reaction of CF0F1. Experimental and theoretical deuterium isotope effects of the localized hydrolysis on the activities of CF0F1, and the energetics of these related reactions, support this proposed mechanism. Our experimental observations could be understood in the framework of the well-established ion translocation models for the H+-ATP synthase from Escherichia coli, and the Na+-ATP synthase from Propionigenium modestum and Ilyobacter tartaricus.  相似文献   

16.
This study investigated the effects of tetracycline on photophosphorylation, electron transport and P/O ratio of spinach chloroplasts. When chloroplast preparations were treated with low concentrations of tetracycline, non-cyclic and cyclic photophosphorylation activities increased, electron transport rates and P/O ratios improved, chloroplast ms-DLE also improved, and the Mg2+-ATPase activity of CF1 increased in comparison to the control. These results indicate that spinach chloroplasts are sensitive to tetracycline. Next, we used the fluorescence emission spectra of CF1 to examine the possible binding sites for tetracycline. The fluorescence emission spectra of CF1 treated with glutaraldehyde, NEM and TNBS, which interact with CF1 across its whole structure, at the γ subunit and at the β subunit, respectively, were compared with that of control CF1. The peak sites of the various fluorescence emission spectra were the same, but the peak values for CF1 treated with glutaraldehyde, NEM and TNBS were lower than that of control CF1. The peak value of CF1 treated with 50 μM tetracycline was very similar to that of CF1 treated with NEM. The above results indicate that the acting site of tetracycline may be at or near the γ subunit of CF1, and allows the creation of a model in which tetracycline binding strengthens the subunit interactions of ATP synthase, enlarges the proton motive force across the thylakoid membrane, and allows the excess proton motive force to increase ATP formation and improve the P/O ratio. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

17.
F1-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F0 to synthesize ATP in vivo. Therefore elucidation of the behavior of F1 under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F1 rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 μM), 120° stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque.  相似文献   

18.
The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF0CF1 ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg2+ and strong thylakoid stacking in the presence of only low Mg2+ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF1 and the CF0CF1 complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented.  相似文献   

19.
The FoF1 synthase produces ATP from ADP and inorganic phosphate. The γ subunit of FoF1 ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole FoF1 complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire FoF1 ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of FoF1 ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of FoF1 ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.  相似文献   

20.
Summary The proton-driven ATP synthase of chloroplasts is composed of two elements, CF0 and CF1. The membrane bound CF0 conducts protons and the peripheral CF1 interacts with nucleotides. By flash spectrophotometric techniques applied to thylakoid membranes from which about 50% of total CF1 was removed, we have previously determined the protonic (timeaveraged) single-channel conductance of CF0. Being in the order of 1 pS, it was sufficiently large to support the proposed role of CF0 as a low-impedance access for protons to the coupling site in CF0CF1. On the other hand, it was too large to be readily reconciled with current concepts of proton supply to and proton conduction through the channel.We studied the time-averaged single-channel conductance of CF0 under variation of pH, pD, ionic composition, temperature, and water/membrane structure with the following results: (i) CF0 was proton-specific even against a background of 300mm monovalent or 30mm divalent catins. (ii) While the conductance of CF0 was pH/pD-independent in the range from 5.6–8.0, in D2O it was lower by a constant factor of 1.7 than in H2O (iii) Addition of glycerol diminished the conductance and abolished the isotope effect. (iv) The Arrhenius activation energy was 42 kJ/mol and thus intermediate between the ones found for the water-filled pore, gramicidin (30 kJ/mol), and the mobile carrier, valinomycin (65 kJ/mol).The results implied that CF0 is endowed with an extremely proton-specific (107-fold) selectivity filter. Its conductance is very high, and its conduction cycle is not necessarily rate limited by a protolytic reaction. The mechanisms of rapid proton supply to the channel mouth and of proton conduction remained enigmatic.  相似文献   

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