Search

Your search keyword '"Sigalat C"' showing total 6 results
Start Over Author "Sigalat C" Search Limiters Full Text
6 results on '"Sigalat C"'

1. The binding mechanism of the yeast F1-ATPase inhibitory peptide. Role of catalytic intermediates and enzyme turnover

Author

Corvest, V., Sigalat, C., Venard, R., Falson, P., Mueller, Dm, Haraux, F., Protéines membranaires transductrices d'énergie (PMTE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Schont, Francoise, Deleage, Gilbert, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
Adenosine Triphosphatases, Binding Sites, Time Factors, Dose-Response Relationship, Drug, Hydrolysis, Tryptophan, Proteins, Saccharomyces cerevisiae, Biochemistry, Models, Biological, Catalysis, Adenosine Diphosphate, Kinetics, Adenosine Triphosphate, Spectrometry, Fluorescence, Models, Chemical, Catalytic Domain, Escherichia coli, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Binding
Abstract

International audience; The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.

Published
2005

2. Mechanism of activation of the chloroplast ATP synthase. A kinetic study of the thiol modulation of isolated ATPase and membrane-bound ATP synthase from spinach by Eschericia coli thioredoxin.

Author

He, X, Miginiac-Maslow, M, Sigalat, C, Keryer, E, and Haraux, F

Abstract

The mechanism of thiol modulation of the chloroplast ATP synthase by Escherichia coli thioredoxin was investigated in the isolated ATPase subcomplex and in the ATP synthase complex reconstituted in bacteriorhodopsin proteoliposomes. Thiol modulation was resolved kinetically by continuously monitoring ATP hydrolysis by the isolated subcomplex and ATP synthesis by proteoliposomes. The binding rate constant of reduced thioredoxin to the oxidized ATPase subcomplex devoid of its epsilon subunit could be determined. It did not depend on the catalytic turnover. Reciprocically, the catalytic turnover did not seem to depend on thioredoxin binding. Thiol modulation by Trx of the epsilon-bearing ATPase subcomplex was slow and favored the release of epsilon. The rate constant of thioredoxin binding to the membrane-bound ATP synthase increased with the protonmotive force. It was lower in the presence of ADP than in its absence, revealing a specific effect of the ATP synthase turnover on thioredoxin-gamma subunit interaction. These findings, and more especially the comparisons between the isolated ATPase subcomplex and the ATP synthase complex, can be interpreted in the frame of the rotational catalysis hypothesis. Finally, thiol modulation changed the catalytic properties of the ATP synthase, the kinetics of which became non-Michaelian. This questions the common view about the nature of changes induced by ATP synthase thiol modulation.

Published
2000

3. Interrelation between high and low affinity tentoxin binding sites in chloroplast F1-ATPase revealed by synthetic analogues.

Author

Santolini, J, Haraux, F, Sigalat, C, Munier, L, and André, F

Abstract

Eight synthetic analogues of tentoxin (cyclo-(L-N-MeGlu1-L-Leu2-N-MeDeltaZPhe3-Gly4)) modified in residues 1, 2, and 3 were checked for their ability to inhibit and reactivate the ATPase activity of the activated soluble part of chloroplast ATP synthase. The data were consistent with a model involving two binding sites of different affinities for the toxins. The occupancy of the high affinity site (or tight site) gave rise to an inactive complex, whereas filling both sites (tight + loose) gave rise to a complex of variable activity, dependent on the toxin analogue. Competition experiments between tentoxin and nonreactivating analogues allowed discrimination between the absence of binding and a nonproductive binding to the site of lower affinity (or loose site). The affinity for the loose site was not affected significantly by the modifications of the tentoxin molecule, whereas the affinity for the tight site was found notably changed. Increasing the size of side chain 1 or 2 and introducing a net electrical charge both resulted in a decrease of affinity for the tight site, but the second change dominated the first one. The activity of different ternary complexes enzyme-tentoxin-analogue depended on the nature of the toxin bound on each site and not only on that bound on the loose site. This demonstrates that the reactivation process results from an interaction, direct or not, between these two binding sites. Possible molecular mechanisms are discussed.

Published
1998

4. Kinetic analysis of tentoxin binding to chloroplast F1-ATPase. A model for the overactivation process.

Author

Santolini, J, Haraux, F, Sigalat, C, Moal, G, and André, F

Abstract

The mechanism of action of tentoxin on the soluble part (chloroplast F1 H+-ATPase; CF1) of chloroplast ATP synthase was analyzed in the light of new kinetic and equilibrium experiments. Investigations were done regarding the functional state of the enzyme (activation, bound nucleotide, catalytic turnover). Dialysis and binding data, obtained with 14C-tentoxin, fully confirmed the existence of two tentoxin binding sites of distinct dissociation constants consistent with the observed Kinhibition and Koveractivation. This strongly supports a two-site model of tentoxin action on CF1. Kinetic and thermodynamic parameters of tentoxin binding to the first site (Ki = 10 nM; kon = 4.7 x 10(4) s-1.M-1) were determined from time-resolved activity assays. Tentoxin binding to the high affinity site was found independent on the catalytic state of the enzyme. The analysis of the kinetics of tentoxin binding on the low affinity site of the enzyme showed strong evidence for an interaction between this site and the nucleotide binding sites and revealed a complex relationship between the catalytic state and the reactivation process. New catalytic states of CF1 devoid of epsilon-subunit were detected: a transient overstimulated state, and a dead end complex unable to bind a second tentoxin molecule. Our experiments led to a kinetic model for the reactivation phenomenon for which rate constants were determined. The implications of this model are discussed in relation to the previous mechanistic hypotheses on the effect of tentoxin.

Published
1999

6. The binding mechanism of the yeast F1-ATPase inhibitory peptide: role of catalytic intermediates and enzyme turnover.

Author

Corvest V, Sigalat C, Venard R, Falson P, Mueller DM, and Haraux F

Subjects
Adenosine Diphosphate chemistry, Adenosine Triphosphatases chemistry, Adenosine Triphosphate chemistry, Binding Sites, Biochemistry methods, Catalysis, Catalytic Domain, Dose-Response Relationship, Drug, Escherichia coli metabolism, Hydrolysis, Kinetics, Models, Biological, Models, Chemical, Protein Binding, Proteins metabolism, Saccharomyces cerevisiae metabolism, Spectrometry, Fluorescence, Time Factors, Tryptophan chemistry, ATPase Inhibitory Protein, Proteins chemistry
Abstract

The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.

Published
2005
Full Text
View/download PDF

Catalog

Books, media, physical & digital resources
See catalog results