Kinetic analysis of tentoxin binding to chloroplast F-1-ATPase - A model for the overactivation process

The mechanism of action of tentoxin on the soluble part (chloroplast F-1 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 tu rnover). Dialysis and binding data, obtained with C-14-tentoxin, fully confirmed the existence of two tentoxin binding sites of distinct dissociation constants consistent with the observed K-inhibition and K-overactivation. This stron gly supports a two-site model of tentoxin action on CF1. Kinetic and thermo dynamic parameters of tentoxin binding to the first site (K-i = 10 nM; k(on ) = 4.7 x 10(4) s(-1).M-1) were determined from time-resolved activity assa ys. 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 o f the enzyme showed strong evidence for an interaction between this site an d the nucleotide binding sites and revealed a complex relationship between the catalytic state and the reactivation process. New catalytic states of C F1 devoid of epsilon-subunit were detected: a transient overstimulated stat e, and a dead end complex unable to bind a second tentoxin molecule. Our ex periments led to a kinetic model for the reactivation phenomenon for which rate constants were determined. The implications of this model are discusse d in relation to the previous mechanistic hypotheses on the effect of tento xin.