<i>Escherichia coli</i> ATP Synthase α Subunit Arg-376:  The Catalytic Site Arginine Does Not Participate in the Hydrolysis/Synthesis Reaction but Is Required for Promotion to the Steady State

Le, Nga Phi; Omote, Hiroshi; Wada, Yoh; Al‐Shawi, Marwan K.; Nakamoto, Robert K.; Futai, Masamitsu

doi:10.1021/bi992530h

Cited by 47 publications

(43 citation statements)

References 41 publications

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“…Analyzing mutant enzymes with Lys and Cys at position 376 of the a subunit, we concluded that aArg376 does not play a catalytic role, but is important for conformational transmission among the three sites, as discussed in detail previously (5,29). bGlu185 located near aArg376 in the catalytic site is also close to the phosphate moiety of ATP in the crystal structure (17).…”

Section: Mutational Studies On F-atpasesupporting

confidence: 59%

Rotating proton pumping ATPases: Subunit/subunit interactions and thermodynamics

2013

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In this article, we discuss single molecule observation of rotational catalysis by E. coli ATP synthase (F-ATPase) using small gold beads. Studies involving a low viscous drag probe showed the stochastic properties of the enzyme in alternating catalytically active and inhibited states. The importance of subunit interaction between the rotor and the stator, and thermodynamics of the catalysis are also discussed. ''Single Molecule Enzymology'' is a new trend for understanding enzyme mechanisms in biochemistry and physiology. V C 2013 IUBMB Life, 65(3): [247][248][249][250][251][252][253][254] 2013

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Section: Mutational Studies On F-atpasesupporting

confidence: 59%

Rotating proton pumping ATPases: Subunit/subunit interactions and thermodynamics

2013

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“…38 but see also ref. 39). This difference between the calculated contribution and that obtained from the mutation studies could be caused by several factors (13).…”

Section: Resultsmentioning

confidence: 79%

The missing link between thermodynamics and structure in F ₁ -ATPase

Yang

Gao

Cui

et al. 2003

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

107

106

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F1Fo-ATP synthase is the enzyme responsible for most of the ATP synthesis in living systems. The catalytic domain F 1 of the F1Fo complex, F1-ATPase, has the ability to hydrolyze ATP. A fundamental problem in the development of a detailed mechanism for this enzyme is that it has not been possible to determine experimentally the relation between the ligand binding affinities measured in solution and the different conformations of the catalytic ␤ subunits (␤TP, ␤DP, ␤E) observed in the crystal structures of the mitochondrial enzyme, MF 1. Using free energy difference simulations for the hydrolysis reaction ATP؉H 2O 3 ADP؉Pi in the ␤TP and ␤DP sites and unisite hydrolysis data, we are able to identify ␤TP as the ''tight'' (K D ‫؍‬ 10 ؊12 M, MF1) binding site for ATP and ␤DP as the ''loose'' site. An energy decomposition analysis demonstrates how certain residues, some of which have been shown to be important in catalysis, modulate the free energy of the hydrolysis reaction in the ␤TP and ␤DP sites, even though their structures are very similar. Combined with the recently published simulations of the rotation cycle of F 1-ATPase, the present results make possible a consistent description of the binding change mechanism of F 1-ATPase at an atomic level of detail. T he enzyme F 1 F o -ATP synthase is responsible for most of the ATP synthesis in living systems (1-3). It is a large multisubunit complex consisting of a proton-translocating membrane domain F o attached via central and peripheral stalks to the catalytic domain F 1 , a spherical globular structure outside of the membrane (4-6). The F 1 domain, called F 1 -ATPase, is made up of 3␣ and 3␤ subunits arranged in alternation around the ␣-helical coiled-coil structure of the ␥ subunit. The foot of the ␥ subunit is a more globular domain and makes extensive contacts with the ring of c subunits of the membrane portion, F 0 (7). The ␥ subunit and the associated c ring are believed to rotate as an ensemble relative to the rest of the enzyme, the rotation being generated by the transmembrane proton-motive force via photosynthesis or respiration. The ␣-helical domain of the ␥ subunit is asymmetric and the rotation of this asymmetrical structure alters the conformations (4-6) and the binding affinities (8, 9) of the three catalytic ␤ subunits for substrate and products. Each of them in turn is thought to go through three states known as open, loose, and tight (4), in accord with the ''binding change'' mechanism of ATP synthesis (1). The F 1 domain can be separated from the membrane domain and it retains the ability to hydrolyze ATP. Hydrolysis of ATP leads to the rotation of the central stalk, although the detailed mechanism is not understood. By attaching an actin filament or a bead to the exposed foot of the central stalk, the rotation has been visualized in a microscope (10). The actin filament turns counterclockwise (as viewed from the membrane) in 120°steps. During the ATP synthesis cycle, the rotation of the central stalk is presumed to be in the opposite sense.Of...

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“…Le et al [63] also showed that αArg376 can be replaced and the F 1 enzyme retains the ability to carry out uni-site catalysis; however, the enzyme had essentially no cooperative steady state ATPase activity which involves all three sites. These results suggest that αArg376 may also be important to create the high affinity Pi binding site as β E converts to β DP during ATP synthesis (Fig.…”

Section: Partial Reaction and Rotation Steps In Steady Statementioning

confidence: 99%

The rotary mechanism of the ATP synthase

Nakamoto

Scanlon

Al‐Shawi

2008

Archives of Biochemistry and Biophysics

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167

126

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The F O F 1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous F O sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium. KeywordsATP synthase; kinetic mechanism; rotation; transport Like many transporters, the F O F 1 ATP synthase (or F-type ATPase) has been a fascinating subject for the study of a complex membrane-associated process. The ATP synthase is a critically important activity that carries out synthesis of ATP from ADP and Pi driven by a proton motive force, Δµ H+ , or sodium motive force, Δµ Na+ . This final step of oxidative or photo-phosphorylation provides the vast majority of ATP in the cell. The proton or sodium motive force is also needed to power other membrane processes such as secondary transporters or in the case of bacteria, flagellum rotation. In anaerobic conditions, facultative bacteria use the ATP synthase as an ATP-driven H + or Na + pump to generate the Δµ H+ , or Δµ Na+ (see [1] for a textbook review.) The F O F 1 complex is nearly ubiquitous in the cell membranes of eubacteria, in the thylakoid membrane of chloroplasts, and the inner membrane of mitochondria. The transporter has remained structurally and mechanistically conserved, except for a few additional domains or subunits in mitochondria, which may play roles in regulation or assembly.Many years of innovative biochemical, genetic, kinetic, and thermodynamic studies led to the first structural solution of the catalytic F 1 portion of the complex by Walker, Leslie and coworkers [2] in 1994. This landmark structure provided critical information on the catalytic portion of the complex but the subunit arrangement of much of the rest of the complex was still not elucidated. The partial F 1 structure, which at the time was the largest asymmetric unit solved, provided the impetus and the structural information needed to test the notion that the

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Escherichia coli ATP Synthase α Subunit Arg-376: The Catalytic Site Arginine Does Not Participate in the Hydrolysis/Synthesis Reaction but Is Required for Promotion to the Steady State

Cited by 47 publications

References 41 publications

Rotating proton pumping ATPases: Subunit/subunit interactions and thermodynamics

Rotating proton pumping ATPases: Subunit/subunit interactions and thermodynamics

The missing link between thermodynamics and structure in F ₁ -ATPase

The rotary mechanism of the ATP synthase

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Escherichia coli ATP Synthase α Subunit Arg-376: The Catalytic Site Arginine Does Not Participate in the Hydrolysis/Synthesis Reaction but Is Required for Promotion to the Steady State

Cited by 47 publications

References 41 publications

Rotating proton pumping ATPases: Subunit/subunit interactions and thermodynamics

Rotating proton pumping ATPases: Subunit/subunit interactions and thermodynamics

The missing link between thermodynamics and structure in F 1 -ATPase

The rotary mechanism of the ATP synthase

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The missing link between thermodynamics and structure in F ₁ -ATPase