Reactions related with Δũ H +-dependent

Abstract

The process of Δgm H + -fdependent activation of the chloroplast H + -ATPase was studied by following initial kinetics oflight-induced release of tightly bound labeled nucleotides from CF, of pre-loaded thylakoids. The following results were obtained. (1) The initial rate of light-induced nucleotide release is increased by medium ADP and further accelerated by the simultaneous presence of medium phosphate. In the latter case a biphasic time-course is observed, indicating a fast activation of up to 30% of the ATPase followed by a slow activation of the rest. The rapid initial phase is absent when the medium contains ADP only. Phosphate alone does not stimulate light-induced nucleotide release. (2) During the first 20–30 ms of illumination in the presence of ADP and P, the rates of nucleotide release and photophosphorylation are identical, suggesting a close relationship between the two processes. The initial rates of both reactions are strongly inhibited by valinomycin (in presence of K + ), indicating the significance of a membrane potential as the driving force. In particular the initial ADP + P i -stimulated nucleotide release is inhibited by DCCD. The tightly bound ADP found in deenergized thylakoids is not the initial phosphoryl acceptor in photophosphorylation. (3) Omission or replacement of Mg 2+ by Ca 2+ and substitution of phosphate by phosphate analogues permits the conclusion that maximal stimulation of light-induced nucleotide release requires binding of P i and ADP-Mg rather than ATP formation at other sites of the enzyme. (4) The effect of varying medium ADP and P i concentrations on the initial velocity of light-induced nucleotide release reveals two different substrate constants for each of the two compounds. The results suggest participation of two functionally linked free centers in acceleration of nucleotide release. Successive occupation of these two centers with ADP and P i executes a negative cooperative effect on the third, nucleotide-containing site. The above-mentioned results are explained on the basis of a model employing three principally identical catalytic centers which are occluded in the inactive state of the enzyme. One of them is occupied with ADP or ATP, two are non-occupied. Opening of the three centers by Δũ H + -linked conformational changes renders nucleotide dissociation and interaction of medium substrates with the free sites. Binding of ADP and P i , and simultaneous forced dissociation of the former tightly bound nucleotide are regarded as predisposing steps to establish the functional order of the sites involved in catalytic turnover.