ROTATIONAL COUPLING IN THE F 0 F 1 ATP SYNTHASE

Self Cite

The temperature-dependent rotation of F 1 -ATPase ␥ subunit was observed in V max conditions at low viscous drag using a 60-nm gold bead (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126 -4131). The Arrhenius slopes of the speed of the individual 120°steps and reciprocal of the pause length between rotation steps were very similar, indicating a flat energy pathway followed by the rotationally coupled catalytic cycle. In contrast, the Arrhenius slope of the reciprocal pause length of the ␥M23K mutant F 1 was significantly increased, whereas that of the rotation rate was similar to wild type. The effects of the rotor ␥M23K substitution and the counteracting effects of ␤E381D mutation in the interacting stator subunits demonstrate that the rotor-stator interactions play critical roles in the utilization of stored elastic energy. The ␥M23K enzyme must overcome an abrupt activation energy barrier, forcing it onto a less favored pathway that results in uncoupling catalysis from rotation.

Section: ␤Delseedsupporting

confidence: 80%

Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of γ-β Subunit Interactions in the Catalytic Dwell

Sekiya

Nakamoto

Al‐Shawi

et al. 2009

Self Cite

“…So far, we failed to detect MgADP inhibition in E. coli F 1 under the conditions used to measure MgATP hydrolysis. 2 It is nevertheless important to note that for enzymes and/or under conditions where MgADP inhibition can be observed, MgIDP inhibition is much less pronounced or absent (32)(33)(34). The ITPase assays reported in this work showed no deviations from linearity at any [ITP].…”

Section: Conditionsmentioning

confidence: 69%

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

Weber

Senior

2001

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...

“…The ␥ and ⑀ subunits form the central stalk, rotation of which is believed to be caused by translocation of protons across the membrane by the a and c subunits. This rotation is thought to cause conformational changes in the catalytic sites, driving synthesis of ATP (1). It is believed that the ␣ and ␤ subunits are prevented from rotating by a peripheral stalk consisting of ␦ and the two b subunits (5,6) that joins the ␣ 3 ␤ 3 complex to the a subunit.…”

mentioning

confidence: 99%

“…Exactly how the b subunits interact with a and/or c is also unknown, but it is generally believed that the transmembrane domains of the b subunits lie on the outside of the ring of c subunits (1,21). Chemical cross-linking of purified ATP synthase (22) or F 0 (23) indicates that a and b are proximal, and analysis of second-site revertants to a bG9D mutation identified residue 240 of the a subunit, within the fifth putative transmembrane helix (24,25), as a site that may be close to b within the membrane (26).…”

mentioning

confidence: 99%

Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase

McLachlin¹,

Coveny

Clark

et al. 2000

The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to act as a stator to hold the ␣ 3 ␤ 3 hexamer stationary relative to the a subunit as the ␥⑀c 9 -12 complex rotates. Despite their essential nature, the contacts between b and the ␣, ␤, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b 24 -156 , a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F 1 sector or to complete ATP synthase, or F 1 F 0 -ATPase, utilizes a transmembrane proton gradient to synthesize ATP and is responsible for the final step in oxidative phosphorylation and photophosphorylation. The enzyme (reviewed in Refs. 1-3) is composed of two sectors. The membrane-integral F 0 sector is a proton pore, and in Escherichia coli has a subunit composition of ab 2 c 9 -12 . The membrane-peripheral F 1 sector has a subunit stoichiometry of ␣ 3 ␤ 3 ␥␦⑀. A key feature of the F 1 sector, as seen in the bovine heart mitochondrial crystal structure (4), is that the ␣ and ␤ subunits alternate in a ring around a lengthy pair of ␣-helices of ␥. Each ␤ subunit bears one catalytic nucleotide-binding site, while non-catalytic nucleotide-binding sites are found on the ␣ subunits. These nucleotide-binding sites are located close to the interfaces between ␣ and ␤ subunits, with one site near each of the six interfaces.Subunits from each sector contribute to the formation of two stalks that join F 1 and F 0 . The ␥ and ⑀ subunits form the central stalk, rotation of which is believed to be caused by translocation of protons across the membrane by the a and c subunits. This rotation is thought to cause conformational changes in the catalytic sites, driving synthesis of ATP (1). It is believed that the ␣ and ␤ subunits are prevented from rotating by a peripheral stalk consisting of ␦ and the two b subunits (5, 6) that joins the ␣ 3 ␤ 3 complex to the a subunit.In recent years the interaction of the b dimer and ␦ has been well established by a variety of evidence (7-10). The ␦ subunit appears to be located near the crown of the F 1 complex, the part of F 1 furthest from the membrane (11-15). Because b has a single membrane-spanning region at its N terminus, the remainder of the subunit must span a distance of over 100 Å to come in contact with ␦. Consistent with this proposed arrangement, the region of interaction between b and ␦ has been localized to the C terminus of b (10, 16). The hydrophilic domain of b by itself is mostly ␣-helical as measured by circular dichroism (17), and an isolated complex composed of ␦ with the hydrophilic domain of b was demonstrated by sedimentation velocity ultracentrifugation to be highly extended (9). The dimerization domain of b, encompassing residues 53-122, was also shown to be highly extended (18). Therefore the b 2...