β Subunit Glu-185 of Escherichia coli H+-ATPase (ATP Synthase) Is an Essential Residue for Cooperative Catalysis

Omote, Hiroshi; Le, Nga Phi; Park, Mi-Yeon; Maeda, Masatomo; Futai, Masamitsu

doi:10.1074/jbc.270.43.25656

Cited by 39 publications

(18 citation statements)

References 31 publications

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“…Similar properties were recently reported for the MgATPase activity of S-carboxymethyl-␤185 EcF 1 (49). This residue is equivalent to MF 1 ␤-E192, which is located at the catalytic nucleotide binding sites of F 1 , whereas the RrF 1 ␤-E195 residue is pointing into the tunnel leading to these sites (8).…”

Section: Discussionsupporting

confidence: 54%

Mutagenesis of β-Glu-195 of the Rhodospirillum rubrum F1-ATPase and Its Role in Divalent Cation-dependent Catalysis

Nathanson

Gromet-Elhanan

1998

Journal of Biological Chemistry

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We introduced mutations at the fully conserved residue Glu-195 in subunit ␤ of Rhodospirillum rubrum F 1 -ATPase. The activities of the expressed wild type (WT) and mutant ␤ subunits were assayed by following their capacity to assemble into the earlier prepared ␤-depleted, membrane-bound R. rubrum enzyme (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8742-8747) and to restore ATP synthesis and/or hydrolysis activity. All three mutations, ␤-E195K, ␤-E195Q, and ␤-E195G, were found to bind as the WT␤ into the ␤-depleted enzyme. They restored between 30 and 60% of the WT restored photophosphorylation activity and 16, 45, and 105%, respectively of the CaATPase activity. The mutants required, however, much higher concentrations of divalent cations and could not restore any significant MgATPase or MnATPase activities. Only ␤-E195G could restore some of these activities when assayed in the presence of 100 mM sulfite and high MgCl 2 or MnCl 2 concentrations. These results suggest that the observed difference in restoration of ATP synthesis and CaATPase, as compared with MgATPase and MnATPase, can be due to the tight regulation of the last two activities, resulting in their inhibition at cation/ATP ratios above 0.5. The R. rubrum F 1 ␤-E195 is equivalent to the mitochondrial F 1 ␤-E199, which points into the tunnel leading to the F 1 catalytic nucleotide binding sites (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). Our findings indicate that this residue, although not an integral part of the F 1 catalytic sites, affects divalent cation binding and release of inhibitory MgADP, suggesting its participation in the interconversion of the F 1 catalytic sites between different conformational states.All respiratory and photosynthetic cells contain a membrane-embedded F 0 F 1 ATP synthase that generates ATP at the expense of the electrochemical proton gradient formed during electron transport. The catalytic F 1 component of the enzyme has been solubilized as a functional ATPase from many bacterial, mitochondrial, and chloroplast sources. It is a very conserved multimeric assembly with a stoichiometry of ␣ 3 ␤ 3 ␥␦⑀ and has up to six nucleotide binding sites. Three of them are catalytic sites, residing mainly on the ␤ subunits, and three are noncatalytic, located mainly on the ␣ subunits (1-6).Two recently published x-ray crystallographic structures of rat liver mitochondrial MF 1 at 3.6 Å (7) and bovine heart MF 1 at 2.8 Å (8) have confirmed the alternate arrangement of the six large ␣ and ␤ subunits in a closed hexamer. The structure at 2.8 Å resolution provides direct proof for the earlier suggested location of all six nucleotide binding sites at ␣/␤ interfaces (see Ref. 9). It has also resolved two long C-and Nterminal helical domains of the ␥ subunit, which are embedded within the internal cavity of the ␣ 3 ␤ 3 hexamer, and impose on it an asymmetric structure. This asymmetry is also reflected in different conformational states displayed by the thre...

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Section: Discussionsupporting

confidence: 54%

Mutagenesis of β-Glu-195 of the Rhodospirillum rubrum F1-ATPase and Its Role in Divalent Cation-dependent Catalysis

Nathanson

Gromet-Elhanan

1998

Journal of Biological Chemistry

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“…Whether the inhibitory Mg 2+ is liganded in part to the tightly bound ADP or is bound elsewhere on the enzyme remains uncertain. Mutational replacements of E. coli βGlu-185 gave evidence that this residue, near the catalytic site, was essential for cooperative catalysis and may participate in Mg 2+ binding (121). The ATPase activity of CF 1 and of chloroplast thylakoids is more readily inhibited than the mitochondrial enzyme.…”

Section: Kinetic Evaluation Of Catalytic Site Occupancymentioning

confidence: 99%

The Atp Synthase—a Splendid Molecular Machine

Boyer

1997

Annu. Rev. Biochem.

1,840

1,448

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An X-ray structure of the F1 portion of the mitochondrial ATP synthase shows asymmetry and differences in nucleotide binding of the catalytic beta subunits that support the binding change mechanism with an internal rotation of the gamma subunit. Other structural and mutational probes of the F1 and F0 portions of the ATP synthase are reviewed, together with kinetic and other evaluations of catalytic site occupancy and behavior during hydrolysis or synthesis of ATP. Subunit function as related to proton translocation and rotational catalysis is considered. Physical demonstrations of the gamma subunit rotation have been achieved. The findings have implications for other enzymatic catalyses.

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“…The mechanism underlying the inhibition is worth considering. One possibility is that much of the enzyme is inactive because of the binding of the inhibitory MgADP at the active site (41,42). It is also possible that both active and inactive conformations of F 1 may exist in a slow dynamic equilibrium.…”

Section: Discussionmentioning

confidence: 99%

Stochastic High-speed Rotation of Escherichia coli ATP Synthase F1 Sector

Nakanishi‐Matsui

Kashiwagi

Hosokawa

et al. 2006

Journal of Biological Chemistry

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The ␥ subunit of the ATP synthase F 1 sector rotates at the center of the ␣ 3 ␤ 3 hexamer during ATP hydrolysis. A gold bead (40 -200 nm diameter) was attached to the ␥ subunit of Escherichia coli F 1 , and then its ATP hydrolysis-dependent rotation was studied. The rotation speeds were variable, showing stochastic fluctuation. The high-speed rates of 40-and 60-nm beads were essentially similar: 721 and 671 rps (revolutions/s), respectively. The average rate of 60-nm beads was 381 rps, which is ϳ13-fold faster than that expected from the steady-state ATPase turnover number. These results indicate that the F 1 sector rotates much faster than expected from the bulk of ATPase activity, and that ϳ10% of the F 1 molecules are active on the millisecond time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate/s), as a single molecule, is variable during a short period. The ⑀ subunit inhibited rotation and ATPase, whereas ⑀ fused through its carboxyl terminus to cytochrome b 562 showed no effect. The ⑀ subunit significantly increased the pausing time during rotation. Stochastic fluctuation of catalysis may be a general property of an enzyme, although its understanding requires combining studies of steady-state kinetics and single molecule observation.The biological energy currency ATP is synthesized by a ubiquitous ATP synthase (F 0 F 1 ) conserved in mitochondria, chloroplasts, and bacteria (see Refs. 1-5, for reviews). Escherichia coli F 0 F 1 has a basic subunit structure consisting of catalytic F 1 (␣ 3 ␤ 3 ␥␦⑀) and transmembrane F 0 (ab 2 c 10 ) sectors with a distinct stoichiometry of eight subunits. The ␣ and ␤ subunits form a catalytic hexamer, ␣ 3 ␤ 3 , the ␥ subunit being located at its center. Ten molecules of the c subunit form a membraneembedded ring for continuous proton transport through its interface with subunit a (6 -8). ATP at a catalytic site in each ␤ subunit is synthesized or hydrolyzed cooperatively, as predicted by the binding change mechanism or rotational catalysis (2), and supported by the crystal structure of the ␣ 3 ␤ 3 ␥ complex (9). The ␥ subunit rotation has been shown biochemically by photobleaching of a probe attached to the carboxyl terminus of the ␥ subunit (10), chemical cross-linking between the ␤ and ␥ subunits (11), cryoelectron microscopy (12), and finally video recording of the ATP-dependent anti-clockwise rotation of an actin filament attached to the ␥ subunit (13-15). Consistent with tight interaction between the ␥ and ⑀ subunits, a filament connected to ⑀ rotated together with the ␥ subunit (16).For proton translocation, rotation of the ␥ subunit is transmitted to the c subunit ring, as shown by rotation of the ␥⑀c 10 complex relative to the ␣ 3 ␤ 3 ab 2 (17). Mechanical rotation of purified F 0 F 1 (17-21) and its membrane-bound form have been shown experimentally (22, 23). The rotation of ␥⑀c 10 was consistent with cross-linking experiments (21, 24 -26). The ␥ subunit and ␥⑀c 10 rotated in F 1 and F 0 F 1 , respectively, ...

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β Subunit Glu-185 of Escherichia coli H+-ATPase (ATP Synthase) Is an Essential Residue for Cooperative Catalysis

Cited by 39 publications

References 31 publications

Mutagenesis of β-Glu-195 of the Rhodospirillum rubrum F1-ATPase and Its Role in Divalent Cation-dependent Catalysis

Mutagenesis of β-Glu-195 of the Rhodospirillum rubrum F1-ATPase and Its Role in Divalent Cation-dependent Catalysis

The Atp Synthase—a Splendid Molecular Machine

Stochastic High-speed Rotation of Escherichia coli ATP Synthase F1 Sector

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