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

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

Index: J. Biol. Chem. 273(6) , 3343-50, (1998)

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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.