Kinetic analysis of tentoxin binding to chloroplast F1-ATPase. A model for the overactivation process.
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.
Santolini
J
J
Section de Bioénergétique, Département de Biologie Cellulaire et Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette Cedex, France. santo@dsvidf.cea.fr
Haraux
F
F
Sigalat
C
C
Moal
G
G
André
F
F
D002736:Chloroplasts / Q000201:enzymology*; D004789:Enzyme Activation; D007700:Kinetics; D009183:Mycotoxins / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000378:metabolism*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013379:Substrate Specificity
D009183:Mycotoxins; D010456:Peptides, Cyclic; D006180:Proton-Translocating ATPases; C003384:tentoxin