The α3(βMet222Ser/Tyr345Trp)3γ Subcomplex of the TF1-ATPase Does Not Hydolyze ATP at a Significant Rate until the Substrate Binds to the Catalytic Site of the Lowest Affinity

Ren, Huimiao; Bandyopadhyay, Sanjay; Allison, William S.

doi:10.1021/bi060232w

Cited by 19 publications

(5 citation statements)

References 32 publications

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“…Our data are consistent with tri-site ATP hydrolysis, a model supported in all the reports using tryptophan to probe the filling of catalytic sites, using F 1 -ATPase from E. coli (46,51) as well as from Bacillus PS3 (47,48). In contrast, data obtained on bovine F 1 -ATPase by a rapid filtration technique supported bi-site ATP hydrolysis (52), but they were questioned in a later report (53). Fluorescence data from Figures 4 and 5 could actually be fitted using a bi-site model by assuming that the two sites do not equally contribute to βY345 fluorescence (not shown).…”

Section: Discussionsupporting

confidence: 86%

Insight into the Bind−Lock Mechanism of the Yeast Mitochondrial ATP Synthase Inhibitory Peptide

2007

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The mechanism of yeast mitochondrial F1-ATPase inhibition by its regulatory peptide IF1 was investigated with the noncatalytic sites frozen by pyrophosphate pretreatment that mimics filling by ATP. This allowed for confirmation of the mismatch between catalytic site occupancy and IF1 binding rate without the kinetic restriction due to slow ATP binding to the noncatalytic sites. These data strengthen the previously proposed two-step mechanism, where IF1 loose binding is determined by the catalytic state and IF1 locking is turnover-dependent and competes with IF1 release (Corvest, V., Sigalat, C., Venard, R., Falson, P., Mueller, D. M., and Haraux, F. (2005) J. Biol. Chem. 280, 9927-9936). They also demonstrate that noncatalytic sites, which slightly modulate IF1 access to the enzyme, play a minor role in its binding. It is also shown that loose binding of IF1 to MgADP-loaded F1-ATPase is very slow and that IF1 binding to ATP-hydrolyzing F1-ATPase decreases nucleotide binding severely in the micromolar range and moderately in the submillimolar range. Taken together, these observations suggest an outline of the total inhibition process. During the first catalytic cycle, IF1 loosely binds to a catalytic site with newly bound ATP and is locked when ATP is hydrolyzed at a second site. During the second cycle, blocking of ATP hydrolysis by IF1 inhibits ATP from becoming entrapped on the third site and, at high ATP concentrations, also inhibits ADP release from the second site. This model also provides a clue for understanding why IF1 does not bind ATP synthase during ATP synthesis.

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Section: Discussionsupporting

confidence: 86%

Insight into the Bind−Lock Mechanism of the Yeast Mitochondrial ATP Synthase Inhibitory Peptide

2007

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“…The K d value for the binding of Mg⅐ATP to the low affinity catalytic site (12) matches the K m value for Mg⅐ATP hydrolysis (18,19) and the K m value for rotation in the direct observation of single F 1 molecules (20). Rotational catalysis requires the concerted, sequential participation of all three catalytic sites as they pass through the three different conformations (18,21,22). For these reasons, we have argued (23) that reversible hydrolysis/synthesis of ATP occurs in the ␤ TP site, and product P i and ADP are released after the site converts from ␤ DP to ␤ E (18).…”

mentioning

confidence: 64%

A Rotor-Stator Cross-link in the F1-ATPase Blocks the Rate-limiting Step of Rotational Catalysis

Scanlon

Al‐Shawi

Nakamoto

2008

Journal of Biological Chemistry

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The F 0 F 1 -ATP synthase couples the functions of H ؉ transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located ␥, ⑀, and c subunits. To understand the ␥ subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F 1 -ATPase enzyme that uniquely fits the pre-steady state and steady state data (Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797). Here we directly test the model using two single cysteine mutants, ␤D380C and ␤E381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved ␥Cys-87. In the pre-steady state, the ␥-␤ cross-linked enzyme at high Mg⅐ATP conditions retained the burst of hydrolysis but was not able to release P i . These data show that the rate-limiting rotation step, k ␥ , occurs after hydrolysis and before P i release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the ␤Glu-381 residue for proper formation of the rate-limiting transition state involving ␥ subunit rotation.

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“…After 1 h of incubation at 23°C, the ␣ 3 ␤ 3 ␥ subcomplex was passed through two subsequent centrifuge columns containing Sephadex G-50, equilibrated with 50 mM Tris/ HCl and 0.1 mM EDTA, pH 8.0. After this treatment, the enzyme subcomplex is essentially nucleotide-free (29). Fluorescence titrations were performed in a buffer containing 50 mM Tris/H 2 SO 4 , 2.5 mM MgSO 4 , pH 8.0, with ATP added in the appropriate concentrations.…”

Section: Methodsmentioning

confidence: 99%

The Role of the βDELSEED-loop of ATP Synthase

Mnatsakanyan

Krishnakumar

Suzuki

et al. 2009

Journal of Biological Chemistry

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ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix motif, termed "DELSEED-loop," in the C-terminal domain of the ␤ subunit was suggested to be involved in coupling between catalysis and rotation. Here, the role of the DELSEED-loop was investigated by functional analysis of mutants of Bacillus PS3 ATP synthase that had 3-7 amino acids within the loop deleted. All mutants were able to catalyze ATP hydrolysis, some at rates several times higher than the wild-type enzyme. In most cases ATP hydrolysis in membrane vesicles generated a transmembrane proton gradient, indicating that hydrolysis occurred via the normal rotational mechanism. Except for two mutants that showed low activity and low abundance in the membrane preparations, the deletion mutants were able to catalyze ATP synthesis. In general, the mutants seemed less well coupled than the wild-type enzyme, to a varying degree. Arrhenius analysis demonstrated that in the mutants fewer bonds had to be rearranged during the rate-limiting catalytic step; the extent of this effect was dependent on the size of the deletion. The results support the idea of a significant involvement of the DELSEED-loop in mechanochemical coupling in ATP synthase. In addition, for two deletion mutants it was possible to prepare an ␣ 3 ␤ 3 ␥ subcomplex and measure nucleotide binding to the catalytic sites. Interestingly, both mutants showed a severely reduced affinity for MgATP at the high affinity site.F 1 F 0 -ATP synthase catalyzes the final step of oxidative phosphorylation and photophosphorylation, the synthesis of ATP from ADP and inorganic phosphate. F 1 F 0 -ATP synthase consists of the membrane-embedded F 0 subcomplex, with, in most bacteria, 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, a sodium ion) gradient. Proton flow down the gradient through F 0 is coupled to ATP synthesis on F 1 by a unique rotary mechanism. The protons flow through (half) channels at the interface of the 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 the formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in the 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. 1-6).F 1 (or F 1 -ATPase) has three catalytic nucleotide binding sites loca...

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The α₃(βMet²²²Ser/Tyr³⁴⁵Trp)₃γ Subcomplex of the TF₁-ATPase Does Not Hydolyze ATP at a Significant Rate until the Substrate Binds to the Catalytic Site of the Lowest Affinity

Cited by 19 publications

References 32 publications

Insight into the Bind−Lock Mechanism of the Yeast Mitochondrial ATP Synthase Inhibitory Peptide

Insight into the Bind−Lock Mechanism of the Yeast Mitochondrial ATP Synthase Inhibitory Peptide

A Rotor-Stator Cross-link in the F1-ATPase Blocks the Rate-limiting Step of Rotational Catalysis

The Role of the βDELSEED-loop of ATP Synthase

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