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J Biol Chem, Vol. 274, Issue 2, 849-858, January 8, 1999

Kinetic Analysis of Tentoxin Binding to Chloroplast F₁-ATPase
A MODEL FOR THE OVERACTIVATION PROCESS

Jérôme Santolini, Francis Haraux^¶, Claude Sigalat^¶, Gwénaëlle Moal, and François André^¶

From the ^¶ Protéines Membranaires Transductrices d'Energie (CNRS-URA 2096), DBCM-CEA Saclay, bâtiment 532, F-91191 Gif-sur-Yvette Cedex, France and  Section de Bioénergétique, Département de Biologie Cellulaire et Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette Cedex, France

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 K_inhibition and K_{overactivation}. This strongly supports a two-site model of tentoxin action on CF₁. Kinetic and thermodynamic parameters of tentoxin binding to the first site (K_i = 10 nM; k_on = 4.7 × 10⁴ s¹·M¹) 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 -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.

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