Kinetic Analysis of Tentoxin Binding to Chloroplast F1-ATPase

Santolini, Jérôme; Haraux, Francis; Sigalat, Claude; Moal, Gwénaëlle; André, François

doi:10.1074/jbc.274.2.849

Cited by 17 publications

(45 citation statements)

References 33 publications

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“…At 10 M tentoxin, the time constant for binding was 52 s, whereas the time constant for dissociation was 714 s. The resulting kinetic constants were k on ϭ 1.9 ϫ 10 3 M Ϫ1 s Ϫ1 and k off ϭ 1.4 ϫ 10 Ϫ3 s Ϫ1 . The k on was 1 order of magnitude slower compared with those of the spinach CF 1 , whereas k off was faster than that for CF 1 (10). Nevertheless, binding was fast enough to proceed during rotation experiments, and dissociation was too slow to explain the suppressed rotation.…”

Section: Low Concentrations Of Tentoxin Inhibit the Adp Releasementioning

confidence: 87%

“…During long observation periods, we sometimes found a transition to a slower speed with a single distinct resting position (not shown). For the spinach enzyme, binding and dissociation of stimulatory tentoxin molecule(s) to the binding site(s) take only a few seconds in the presence of ATP (10). We therefore assume that the observed transition was because of release and rebinding of stimulatory tentoxin molecules.…”

Section: Low Concentrations Of Tentoxin Inhibit the Adp Releasementioning

confidence: 99%

“…The purified CF 1 from spinach chloroplasts contains one to two tightly bound ADP molecules (35)(36)(37), and its activity is normally very low because of the ADP inhibition at steady state conditions. The activity of the enzyme can be stimulated more than 10-fold by the addition of high concentrations of the toxin (7)(8)(9)(10). In contrast, purified TF 1 does not contain any bound nucleotides (38,39), and the incubation with Mg-ADP easily induces the strong ADP inhibition state (40).…”

Section: Molecular Processes Of Inhibition and Stimulationmentioning

confidence: 99%

“…However, which of the catalytic processes is affected could not be determined in detail so far. The stimulatory effect of tentoxin is even less understood on the molecular level, although it is accepted that binding of a second or maybe third toxin molecule induces the stimulation (9,10). Previous single molecule experiments using genetically engineered F 1 from thermophilic Bacillus PS3 (TF 1 ) suggest that the high stimulatory concentrations of the toxin correlate with a reduced inhibition of the enzyme by ADP (12).…”

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confidence: 99%

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Molecular Processes of Inhibition and Stimulation of ATP Synthase Caused by the Phytotoxin Tentoxin

Meiß

Konno

Groth

et al. 2008

Journal of Biological Chemistry

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F 1 -ATPase is the smallest mechanical motor known. Tentoxin, a cyclic peptide produced by phytopathogenic fungi, inactivates the F 1 motor in sensitive plants at nanomolar to micromolar concentrations, whereas higher concentrations surpass the natural activity of the enzyme. Single molecule studies now have clarified the molecular steps involved in both processes. Inactivation delays the dwell time of a single step in the complete 360°turn and results in an asymmetric rotation of the central rotor subunit. In contrast, rotation in the stimulated F 1 particle is smooth and accompanied by strongly reduced ADP inhibition. Our study provides for the first time the direct observation of a noncompetitively inhibited state of the enzyme and directly visualizes the regulation of the molecular motor by an external natural compound. In addition, the ADP release step during catalysis was revealed by analysis of the single molecule rotation behavior. Hence, tentoxin is a sophisticated molecular tool to mark and control certain catalytic steps within the reaction pathway of the molecular F 1 motor.

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Section: Low Concentrations Of Tentoxin Inhibit the Adp Releasementioning

confidence: 87%

Section: Low Concentrations Of Tentoxin Inhibit the Adp Releasementioning

confidence: 99%

Section: Molecular Processes Of Inhibition and Stimulationmentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular Processes of Inhibition and Stimulation of ATP Synthase Caused by the Phytotoxin Tentoxin

Meiß

Konno

Groth

et al. 2008

Journal of Biological Chemistry

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“…As a first step toward this goal, we examined the effect of tentoxin on the intrinsic rate of ATP hydrolysis in the absence of a protein import substrate. Tentoxin is a well-characterized inhibitor of the reversible chloroplast CF1/CF0 ATPase responsible for photophosphorylation (18)(19)(20)(21). Whereas the CF1/CF0 ATPase is relatively inactive in dark-adapted chloroplasts (22), it is nonetheless responsible for a low amount of ATP hydrolysis even in its nonactivated form.…”

Section: Effect Of Inhibitors On Intrinsic Background Atpase Activitymentioning

confidence: 99%

Energetic cost of protein import across the envelope membranes of chloroplasts

Shi

Theg

2012

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

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Chloroplasts are the organelles of green plants in which light energy is transduced into chemical energy, forming ATP and reduced carbon compounds upon which all life depends. The expenditure of this energy is one of the central issues of cellular metabolism. Chloroplasts contain ∼3,000 proteins, among which less than 100 are typically encoded in the plastid genome. The rest are encoded in the nuclear genome, synthesized in the cytosol, and posttranslationally imported into the organelle in an energy-dependent process. We report here a measurement of the amount of ATP hydrolyzed to import a protein across the chloroplast envelope membranesonly the second complete accounting of the cost in Gibbs free energy of protein transport to be undertaken. Using two different precursors prepared by three distinct techniques, we show that the import of a precursor protein into chloroplasts is accompanied by the hydrolysis of ∼650 ATP molecules. This translates to a ΔG protein transport of some 27,300 kJ/mol protein imported. We estimate that protein import across the plastid envelope membranes consumes ∼0.6% of the total light-saturated energy output of the organelle. T he majority of proteins in cells are synthesized in the cytoplasm on free or membrane-bound ribosomes. The fraction of proteins that are translocated across one or more cellular membranes has been estimated to be almost 50% in eukaryotic cells (1) and as high as 30% in bacteria (2, 3). Thus, protein movement out of the cytoplasm across membranes represents a considerable cellular activity involving numerous and varied protein transport systems. Protein translocation across membranes is also generally an energy-requiring process, making it a potentially costly activity for cells (4).The nature of the energy inputs to the different protein translocation systems has been extensively studied. We know, for example, that the posttranslational transport of proteins into the endoplasmic reticulum requires ATP hydrolysis alone (4-6), and that the transport of proteins into the thylakoid lumen or across the bacterial plasma membrane on the Tat pathway requires only the transmembrane protonmotive force with no contribution of ATP hydrolysis (7,8). A more complex energy input is required for many other protein transport systems. For instance, the import of proteins into the mitochondrial matrix requires both ATP hydrolysis and the Δψ across the inner mitochondrial membrane (9-11), and bacterial secretion and protein transport to the thylakoid lumen on their respective Sec pathways use ATP hydrolysis and the protonmotive force (3,7,12,13).In contrast to the abundance of work defining the nature of the energy driving protein transporters, few attempts have been made to quantitate the amount of energy required for these reactions. Without this information, it is not possible to assess the amount of metabolic energy that is spent on the cell's considerable protein trafficking activity. One such measurement of the energy required for protein transport is the so-called translo...

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Rebuilt 3D structure of the chloroplast f1 ATPase–tentoxin complex

et al. 2002

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The F1 part of the chloroplast H+ adenosine triphosphate (ATP)-synthase (CF1) strongly interacts with tentoxin, a natural fungous cyclic tetrapeptide known to inhibit the chloroplast enzyme and not the mammalian mitochondrial enzyme. Whereas the synthesis or the hydrolysis of ATP requires the stepwise rotation of the protein rotor gamma within the (alphabeta)3 crown, only one molecule of tentoxin is needed to fully inhibit the complex. With the help of an original homology modeling technique, based on robust distance geometry protocols, we built a tridimensional model of the alpha3beta3gamma CF1) subcomplex (3200 esidues), in which we introduced three different nucleotide occupancies to check their possible influence on the tentoxin binding site. Simultaneous comparison of three available high-resolution X-ray structures of F1, performed with a local structural alignment search tool, led to characterizing common structural blocks and the distorsions experienced by the complex during the catalytic turnover. The common structural blocks were used as a starting point of the spinach CF1 structure rebuilding. Finally, tentoxin was docked into its putative binding site of the reconstructed structure. The docking method was initially validated in the mitochondrial enzyme by its ability to relocate nucleotides into their original position in the crystal. Tentoxin binding was found possible to the two alpha/beta interfaces associated with the empty and adenosine diphosphate (ADP)-loaded catalytic sites, but not to the one associated with the ATP-loaded site. These results suggest a mechanism of CF1 inhibition by one molecule of tentoxin, by the impossibility of the alpha/beta interface bearing tentoxin to pass through the ATP-loaded state.

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Kinetic Analysis of Tentoxin Binding to Chloroplast F1-ATPase

Cited by 17 publications

References 33 publications

Molecular Processes of Inhibition and Stimulation of ATP Synthase Caused by the Phytotoxin Tentoxin

Molecular Processes of Inhibition and Stimulation of ATP Synthase Caused by the Phytotoxin Tentoxin

Energetic cost of protein import across the envelope membranes of chloroplasts

Rebuilt 3D structure of the chloroplast f1 ATPase–tentoxin complex

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