From uni-site to multi-site ATP synthesis in thylakoid membranes

Abstract

The membrane-bound H +-ATPase from chloroplasts, CF 0F 1, was brought into the active, reduced state by illumination in the presence of thioredoxin and dithiothreitol. The endogenous nucleotides were removed by a washing procedure so that the active, reduced enzyme contained one tightly bound ATP per CF 0F 1. When [ 14C]ADP was added in substoichiometric amounts during continuous illumination, ADP was bound to the enzyme, phosphorylated and released as [ 14C]ATP, i.e., the tightly bound ATP was not involved in the catalytic turnover (‘uni-site ATP-synthesis’). The rate constant for ADP binding was k = (2.0 ± 0.5) · 10 6 M −1 s −1. The rate of ATP synthesis was measured as a function of the ADP concentration from 8 nM up to 1 mM in the presence of 2 mM phosphate during continuous illumination. A linear increase of the rate was observed up to 100 nM. Above this concentration a supralinear increase was found, indicating the occupation of a second ADP-binding site. A plateau was reached between 1.5 μM and 2.3 μM ADP with a rate of v pl = 3.7 s −1. The half-maximal rate from this plateau was observed at 780 nM. Above 2.3 μM ADP up to 1 mM ADP the data were described by Michaelis-Menten kinetics ( v max = 80 s −1; apparent K M = 32 μM.) These results indicated the participation of at least two different ADP binding sites in ATP synthesis catalyzed by the membrane-bound CF 0F 1.