Purine but Not Pyrimidine Nucleotides Support Rotation of F1-ATPase

Noji, Hiroyuki; Bald, Dirk; Yasuda, Ryohei; Itoh, Hiroyasu; Yoshida, Masasuke; Kinosita, Kazuhiko

doi:10.1074/jbc.m102200200

Cited by 51 publications

(38 citation statements)

References 19 publications

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“…However, given that (i) MgITP generates ␥ rotation with the same characteristics as MgATP (47), and (ii) that K d3 (MgITP) is Ϸ50-fold higher than K d3 (MgATP) (12,13), it does not seem very likely that the initial step of binding to the low-affinity site drives the rotation substep. Rather, it seems to be the subsequent conversion of low-affinity to high-affinity site, accompanied by a change in conformation of the ␤ subunit from a ''half-closed'' to the fully closed state, which makes ␥ rotate (similar as suggested in ref.…”

Section: Discussionmentioning

confidence: 99%

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Mao

Weber²

2007

Proc. Natl. Acad. Sci. U.S.A.

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ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F1 subcomplex has three catalytic nucleotide binding sites, one on each ␤ subunit, with widely differing affinities for MgATP or MgADP. During rotational catalysis, the sites switch their affinities. The affinity of each site is determined by the position of the central ␥ subunit. The site with the highest nucleotide binding affinity is catalytically active. From the available x-ray structures, it is not possible to discern the high-affinity site. Using fluorescence resonance energy transfer between tryptophan residues engineered into ␥ and trinitrophenyl nucleotide analogs on the catalytic sites, we were able to determine that the high-affinity site is close to the C-terminal helix of ␥, but at considerable distance from its N terminus. Thus, the ␤TP site in the ATP synthase ͉ catalytic mechanism ͉ rotation F 1 F o -ATP synthase is responsible for the bulk of ATP synthesis from ADP and P i in most organisms. F 1 F o -ATP synthase consists of the membrane embedded F o subcomplex with, in Escherichia coli, a subunit composition of ab 2 c 10 , and the peripheral F 1 subcomplex, with a subunit composition of ␣ 3 ␤ 3 ␥␦ . The energy necessary for ATP synthesis is derived from an electrochemical transmembrane proton (or, in some organisms, sodium ion) gradient. Proton flow, down the gradient, through F o is coupled to ATP synthesis on F 1 by a unique rotary mechanism. The protons flow through channels at the interface of a and c subunits, which drives rotation of the ring of c subunits. The c 10 ring, together with F 1 subunits ␥ and , forms the rotor. Rotation of ␥ leads to conformational changes in the catalytic nucleotide binding sites on the ␤ subunits, where ADP and P i are bound. The conformational changes result in formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in form of subunit rotation, and back into chemical energy as ATP. In bacteria, under certain physiological conditions, the process runs in reverse. ATP is hydrolyzed to generate a transmembrane proton gradient which the bacterium requires for such functions as nutrient import and locomotion (for reviews, see refs.

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Section: Discussionmentioning

confidence: 99%

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Mao

Weber²

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…A mutant ␣ 3 ␤ 3 ␥ subcomplex (␣-C193S, ␤-His-10 at N terminus, ␥-S107C͞I210C) from a thermophilic Bacillus PS3 (referred to as F 1 unless otherwise specified) was expressed and purified as described (24). ATP, ADP, and phosphoenolpyruvate were purchased from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Activation of pausing F ₁ motor by external force

Hirono-Hara

Ishizuka

Kinosita

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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102

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A rotary motor F1, a catalytic part of ATP synthase, makes a 120°s tep rotation driven by hydrolysis of one ATP, which consists of 80°a nd 40°substeps initiated by ATP binding and probably by ADP and͞or Pi dissociation, respectively. During active rotations, F1 spontaneously fails in ADP release and pauses after a 80°substep, which is called the ADP-inhibited form. In the present work, we found that, when pushed >؉40°with magnetic tweezers, the pausing F1 resumes its active rotation after releasing inhibitory ADP. The rate constant of the mechanical activation exponentially increased with the pushed angle, implying that F1 weakens the affinity of its catalytic site for ADP as the angle goes forward. This finding explains not only its unidirectional nature of rotation, but also its physiological function in ATP synthesis; it would readily bind ADP from solution when rotated backward by an Fo motor in the ATP synthase. Furthermore, the mechanical work for the forced rotation was efficiently converted into work for expelling ADP from the catalytic site, supporting the tight coupling between the rotation and catalytic event.ADP inhibition ͉ ATP synthase ͉ F1-ATPase ͉ magnetic tweezers ͉ single-molecule observation

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“…Previous studies (17)(18)(19) have indicated that the overall affinities of F 1 for MgGTP and MgITP are considerably lower than those for MgATP, while V max (hydrolysis) appeared to be less affected. Both MgGTP and MgITP are competent at eliciting rotation (19) while MgGDP and MgIDP are good substrates in ATP synthesis (17).…”

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confidence: 99%

“…Both MgGTP and MgITP are competent at eliciting rotation (19) while MgGDP and MgIDP are good substrates in ATP synthesis (17). Unfortunately, GTP has significant absorbance at 295 nm, causing a large inner filter effect in Trp fluorescence experiments.…”

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confidence: 99%

Bi-site Catalysis in F1-ATPase: Does It Exist?

Weber

Senior

2001

Journal of Biological Chemistry

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The mechanism of action of F 1 F 0 -ATP synthase is controversial. Some favor a tri-site mechanism, where substrate must fill all three catalytic sites for activity, others a bi-site mechanism, where one of the three sites is always unoccupied. New approaches were applied to examine this question. First, ITP was used as hydrolysis substrate; lower binding affinities of ITP versus ATP enable more accurate assessment of sites occupancy. Second, distributions of all eight possible enzyme species (with zero, one, two or three sites filled) as fraction of total enzyme population at each ITP concentration were calculated, and compared with measured ITPase activity. Confirming data were obtained with ATP as substrate. Third, we performed a theoretical analysis of possible bi-site mechanisms. The results argue convincingly that bi-site hydrolysis activity is negligible, and may not even exist. Effectively, tri-site hydrolysis is the only mechanism. We argue that only tri-site hydrolysis drives subunit rotation. Theoretical analyses of possible bi-site mechanisms reveal serious flaws, not previously recognized. One is that, in bi-site catalysis, the predicted direction of subunit rotation is the same for both ATP synthesis and hydrolysis; a second is that infrequently occurring enzyme species are required. F 1 is the catalytic sector of ATP synthase, the enzyme that synthesizes ATP in the last step of oxidative phosphorylation (for reviews, see Refs. 1 and 2; short reviews on individual topics can be found in Refs. 3-5). F 1 has a subunit composition of ␣ 3 ␤ 3 ␥␦⑀. The three catalytic nucleotide-binding sites are located at ␣/␤ interfaces, mainly on the ␤ subunits (6). F 1 can be isolated in soluble form and is an active ATPase. It is frequently used as a model for catalysis by the holoenzyme ATP synthase (also called F 1 F 0 ).F 1 (and F 1 F 0 ) can hydrolyze MgATP in different modes, depending on the substrate concentration. Two of these modes are clearly established and well characterized. At low, substoichiometric MgATP concentrations the enzyme binds MgATP with very high affinity, just to catalytic site one. A single turnover of MgATP hydrolysis ensues on this site, called "unisite catalysis," in which the chemical hydrolysis step is slow (0.1 s Ϫ1 in Escherichia coli F 1 , Ref. 1) and products release is even slower (0.001 s Ϫ1 ). On the other hand, at cellular (millimolar) substrate concentrations, all three catalytic sites are filled and interact with each other, and the enzyme turns over rapidly (ϳ100 s Ϫ1 ). This "tri-site" or "multisite catalysis" is the physiologically relevant working mode of the enzyme (1). In multisite catalysis, MgATP hydrolysis on the ␤ subunits drives rotation of the ␥⑀c ensemble (7-9), possibly transmitted via ␣ subunits (10). In contrast, uni-site catalysis can occur without subunit rotation (11).The contribution to hydrolysis of enzyme molecules with two substrate-filled catalytic sites ("bi-site catalysis") remains enigmatic. Originally, deviations from simple Michaelis-Menten kine...

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Purine but Not Pyrimidine Nucleotides Support Rotation of F1-ATPase

Cited by 51 publications

References 19 publications

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Activation of pausing F ₁ motor by external force

Bi-site Catalysis in F1-ATPase: Does It Exist?

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Purine but Not Pyrimidine Nucleotides Support Rotation of F1-ATPase

Cited by 51 publications

References 19 publications

Identification of the β TP site in the x-ray structure of F 1 -ATPase as the high-affinity catalytic site

Identification of the β TP site in the x-ray structure of F 1 -ATPase as the high-affinity catalytic site

Activation of pausing F 1 motor by external force

Bi-site Catalysis in F1-ATPase: Does It Exist?

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Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Activation of pausing F ₁ motor by external force