Catalytic Activity of the α3β3γ Complex of F1-ATPase without Noncatalytic Nucleotide Binding Site

Matsui, Tadashi; Muneyuki, Eiro; Honda, Masahiro; Allison, William S.; Dou, Chao; Yoshida, Masasuke

doi:10.1074/jbc.272.13.8215

Cited by 106 publications

(102 citation statements)

References 47 publications

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“…Binding of ATP to the Isolated Subunits-The kinetics features of inactivation of V 1 -ATPase during the ATPase reaction described above are similar to those recently reported for the mutant F 1 -ATPase, which lacks nucleotide binding at the noncatalytic sites (32). In that case, turnover-dependent inactivation was explained as the failure to recover from the ADP inhibited state due to the inability of nucleotide binding at noncatalytic sites.…”

Section: Kinetics Of Atp Hydrolysis By V 1 -Atpase-because the Atpsupporting

confidence: 64%

V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

Yokoyama

Muneyuki

Amano

et al. 1998

Journal of Biological Chemistry

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The ATP hydrolysis of the V 1 -ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25°C. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 M; from this, an apparent K m of 240 ؎ 24 M and a V max of 5.2 ؎ 0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F 1 -ATPases, was not observed for the V 1 -ATPase. Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V 1 -ATPase was attained by a prior incubation with ADP-Mg. The inactivated V 1 -ATPase contained 1.5 mol of ADP/mol of enzyme.Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit can (3), clathrin-coated vesicles (4), chromaffine granules (5), and the central vacuoles of yeast (6). They are responsible for vacuolar acidification, which plays an important role in a number of cellular processes (1). V o V 1 -ATPases are also found in the plasma membranes of most archea (7-9) and some kinds of eubacteria (10 -12). Several studies indicate that the physiological role of V o V 1 -ATPases of some archea and the thermophilic eubacterium Thermus thermophilus is ATP synthesis coupled to proton flux across the plasma membranes (7,9,(13)(14)(15). Precise understanding of V o V 1 -ATPases would allow the comparison to F o F 1 -ATPases and the elucidation of the common essential mechanism for the coupling of proton translocation across a membrane with ATP formation. However, several problems, such as the difficulty of obtaining a large amount of pure enzyme from vacuolar membranes and an unstable V 1 moiety (17), have limited our investigation of enzymatic properties of V o V 1 -ATPases.T. thermophilus, originally isolated from a hot spring in Japan, is thermophilic, obligatory aerobic, Gram-negative, and chemoheterotrophic eubacterium (25). Its respiratory chain may include energy coupling Site I (26). This bacterium has a large amount of the V o V 1 -ATPase on the plasma membrane, instead of F o F 1 -ATPase (15).In contrast to eukaryotic equivalents, the V 1 moiety of T. thermophilus is easily detached from the membranes using chloroform treatment and ATPase-active stable complex can be obtained in large amounts (10). Throughout this manuscript, the V 1 moiety from T. thermophilus is refereed to V 1 -ATPase.

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Section: Kinetics Of Atp Hydrolysis By V 1 -Atpase-because the Atpsupporting

confidence: 64%

V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

Yokoyama

Muneyuki

Amano

et al. 1998

Journal of Biological Chemistry

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“…Nucleotide-depleted F 1 -ATPase-F 1 -ATPase is known to be inhibited by Mg-ADP tightly bound to a catalytic site (15,16). TF 1 used in the previous study (10) contained ϳ0.3 mol of tightly bound nucleotide/mol of enzyme; thus, the hydrolysis activity might have been underestimated.…”

Section: Resultsmentioning

confidence: 99%

Purine but Not Pyrimidine Nucleotides Support Rotation of F1-ATPase

Noji

Bald

Yasuda

et al. 2001

Journal of Biological Chemistry

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The binding change model for the F 1 -ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120°steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of ϳ40 piconewtons (pN)⅐nm, and mechanical work done in a step of ϳ80 pN⅐nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was ؊80 pN⅐nm/molecule, indicating ϳ100% efficiency. Reconstituted F o F 1 -ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F 1 very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.The F o F 1 ATP synthase is an enzyme that synthesizes ATP from ADP and inorganic phosphate (P i ) using proton flow across a membrane (1). The F o portion of the enzyme resides in the membrane and mediates proton translocation. The F 1 portion, consisting of ␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 subunits, is external to the membrane and catalyzes ATP synthesis. The ATP synthase is a completely reversible molecular machine in that ATP hydrolysis in F 1 can produce a reverse flow of protons through F o . Isolated F 1 only catalyzes ATP hydrolysis and hence is called A crystal structure of F 1 (5) strongly supported the rotation model and has inspired many experiments, which, together, have proved that the ␥ subunit is (part of) the common rotor shaft and that ␣ 3 ␤ 3 subunits, which surrounded ␥ in the crystal, are the stator in the F 1 motor (6 -8). Single-molecule imaging of F 1 , in particular, has revealed that the ␥ subunit rotates in a unique direction consistent with the crystal structure, that ␥ makes discrete 120°steps, and that the energy conversion efficiency of the F 1 motor driven by ATP hydrolysis can reach ϳ100% (9, 10). The precise mechanism of rotation, however, is not yet clear.In Boyer's model, binding changes play the major role (1). On the three ␤ subunits, each of which hosts a catalytic site, ATP and its hydrolysis products, ADP and P i , are in equilibrium. Thus, in the absence of an external energy supply, the change in free energy associated with ATP hydrolysis should manifest as the higher affinity for ATP than for ADP and P i . During ATP synthesis, the mechanical energy supplied by the ␥ rotation driven by F 0 somehow decreases the affinity for A...

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“…60 mM in ¢gure 5). In a mutant where ATP binding to the non-catalytic sites was prohibited, there was no recovery and the inhibition proceeded to completion (Matsui et al 1997). In the assay in ¢gure 5, the hydrolysis products were immediately converted back to ATP.…”

Section: (A) Three Modes Of Hydrolysis Reactionmentioning

confidence: 99%

A rotary molecular motor that can work at near 100% efficiency

Kinosita

Yasuda

Noji

et al. 2000

Phil. Trans. R. Soc. Lond. B

225

141

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A single molecule of F 1 -ATPase is by itself a rotary motor in which a central g-subunit rotates against a surrounding cylinder made of a 3 b 3 -subunits. Driven by the three bs that sequentially hydrolyse ATP, the motor rotates in discrete 1208 steps, as demonstrated in video images of the movement of an actin ¢lament bound, as a marker, to the central g-subunit. Over a broad range of load (hydrodynamic friction against the rotating actin ¢lament) and speed, the F 1 motor produces a constant torque of ca. 40 pN nm. The work done in a 1208 step, or the work per ATP molecule, is thus ca. 80 pN nm. In cells, the free energy of ATP hydrolysis is ca. 90 pN nm per ATP molecule, suggesting that the F 1 motor can work at near 100% e¤ciency. We con¢rmed in vitro that F 1 indeed does ca. 80 pN nm of work under the condition where the free energy per ATP is 90 pN nm. The high e¤ciency may be related to the fully reversible nature of the F 1 motor: the ATP synthase, of which F 1 is a part, is considered to synthesize ATP from ADP and phosphate by reverse rotation of the F 1 motor. Possible mechanisms of F 1 rotation are discussed.

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Catalytic Activity of the α3β3γ Complex of F1-ATPase without Noncatalytic Nucleotide Binding Site

Cited by 106 publications

References 47 publications

V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

Purine but Not Pyrimidine Nucleotides Support Rotation of F1-ATPase

A rotary molecular motor that can work at near 100% efficiency

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