Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase

Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 μM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 μM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional ‘hidden’ Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid.

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On the rate of F1-ATPase turnover during ATP hydrolysis by the single catalytic site Evidence that hydrolysis with a slow rate of product release does not occur at the alternating active site

FEBS Letters ◽

10.1016/0014-5793(87)80186-2 ◽

1987 ◽

Vol 222 (1) ◽

pp. 32-36 ◽

Cited By ~ 17

Author(s):

Ya.M. Milgrom ◽

M.B. Murataliev

Keyword(s):

Active Site ◽

Slow Rate ◽

Atp Hydrolysis ◽

Catalytic Site ◽

F1 Atpase ◽

Product Release

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Steady-state rate of F1 -ATPase turnover during ATP hydrolysis by the single catalytic site

FEBS Letters ◽

10.1016/0014-5793(87)81557-0 ◽

1987 ◽

Vol 212 (1) ◽

pp. 63-67 ◽

Cited By ~ 20

Author(s):

Ya.M. Milgrom ◽

M.B. Murataliev

Keyword(s):

Steady State ◽

Atp Hydrolysis ◽

Catalytic Site ◽

F1 Atpase ◽

Steady State Rate ◽

State Rate

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Kinetics of ATP hydrolysis by F1-ATPase and the effects of anion activation, removal of tightly bound nucleotides, and partial inhibition of the ATPase by covalent modification

Biochemistry ◽

10.1021/bi00316a027 ◽

1984 ◽

Vol 23 (21) ◽

pp. 5004-5009 ◽

Cited By ~ 71

Author(s):

Siu Yin Wong ◽

Akemi Matsuno-Yagi ◽

Youssef Hatefi

Keyword(s):

Atp Hydrolysis ◽

Covalent Modification ◽

F1 Atpase ◽

Partial Inhibition ◽

Kinetics Of ◽

Tightly Bound Nucleotides

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α3β3γ complex of F1-ATPase from thermophilic Bacillus PS3 can maintain steady-state ATP hydrolysis activity depending on the number of non-catalytic sites

Biochemical Journal ◽

10.1042/bj3430135 ◽

1999 ◽

Vol 343 (1) ◽

pp. 135-138 ◽

Cited By ~ 12

Author(s):

Toyoki AMANO ◽

Tadashi MATSUI ◽

Eiro MUNEYUKI ◽

Hiroyuki NOJI ◽

Kiyotaka HARA ◽

...

Keyword(s):

Steady State ◽

Atp Hydrolysis ◽

Catalytic Site ◽

Wild Type ◽

Thermophilic Bacillus ◽

F1 Atpase ◽

Type Complex ◽

Catalytic Sites ◽

Phase Activity ◽

Hydrolysis Activity

Homogeneous preparations of α3β3γ complexes with one, two or three non-competent non-catalytic site(s) were performed as described [Amano, Hisabori, Muneyuki, and Yoshida (1996) J. Biol. Chem. 271, 18128-18133] and their properties were compared with those of the wild-type complex. The ATPase activity of the complex with three non-competent non-catalytic sites decayed rapidly to an inactivated state, as reported previously [Matsui, Muneyuki, Honda, Allison, Dou, and Yoshida (1997) J. Biol. Chem. 272, 8215-8221]. In contrast, the complex with one or two non-competent non-catalytic sites displayed a substantial steady-state phase activity depending on the number of non-competent non-catalytic sites in the complex. This result indicates that one competent non-catalytic site can maintain the continuous catalytic turnover of the enzyme and can potentially relieve all three catalytic sites from inhibition by MgADP-. Furthermore, the results suggest that the interaction between three non-catalytic sites might not be as strong as that between catalytic sites, which are all strictly required for a continuous catalytic turnover.

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Identification of an exchangeable non-catalytic site on mitochondrial F1-ATPase which is involved in the negative cooperativity of ATP hydrolysis

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/0005-2728(93)90161-8 ◽

1993 ◽

Vol 1142 (3) ◽

pp. 327-335 ◽

Cited By ~ 17

Author(s):

C.M. Edel ◽

A.F. Hartog ◽

J.A. Berden

Keyword(s):

Atp Hydrolysis ◽

Catalytic Site ◽

Negative Cooperativity ◽

F1 Atpase

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On the location and function of tyrosine beta 331 in the catalytic site of Escherichia coli F1-ATPase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)46004-8 ◽

1992 ◽

Vol 267 (3) ◽

pp. 1712-1718 ◽

Cited By ~ 5

Author(s):

J Weber ◽

R S Lee ◽

E Grell ◽

J G Wise ◽

A E Senior

Keyword(s):

Escherichia Coli ◽

Catalytic Site ◽

F1 Atpase ◽

And Function

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Specific placement of tryptophan in the catalytic sites of Escherichia coli F1-ATPase provides a direct probe of nucleotide binding: maximal ATP hydrolysis occurs with three sites occupied.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)80703-0 ◽

1993 ◽

Vol 268 (27) ◽

pp. 20126-20133

Author(s):

J Weber ◽

S Wilke-Mounts ◽

R.S. Lee ◽

E Grell ◽

A.E. Senior

Keyword(s):

Escherichia Coli ◽

Atp Hydrolysis ◽

Nucleotide Binding ◽

F1 Atpase ◽

Catalytic Sites

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Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011922 ◽

2019 ◽

Vol 295 (6) ◽

pp. 1551-1564 ◽

Cited By ~ 5

Author(s):

Kelly E. Du Pont ◽

Russell B. Davidson ◽

Martin McCullagh ◽

Brian J. Geiss

Keyword(s):

Molecular Dynamics ◽

Structural Changes ◽

Rna Binding ◽

Atp Hydrolysis ◽

Nonstructural Protein ◽

Binding Pocket ◽

Energy Transduction ◽

Helicase Domain ◽

Genome Replication ◽

Turnover Rates

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential “communication hub” for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

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