The kinetics of photophosphorylation at clamped ΔpH indicate a random order of substrate binding

AbstractThe rate of photophosphorylation of isolated chloroplast thylakoids was investigated at a clamped ΔpH of 2.5 (ΔΨ = 0). On variation of the concentration of ADP at different fixed concentrations of phosphate, straight lines were obtained in double-reciprocal plots which intersected in one point below the x-axis. Upon variation of the phosphate concentration at several fixed concentrations of ADP, similar kinetics were found. These results indicate a random order of binding of the two substrates. Calculation of the dissociation constants and Michaelis constants from these two sets of data according to a bisubstrate rapid equilibrium random model yielded satisfactory agreement. The kinetic constants were found to be unchanged by thiol modulation or demodulation of CF0CF1. The kinetics of inhibition of phosphorylation by the product ATP indicated a competitive effect with regard to ADP as well as phosphate. At a given substrate concentration, the inhibition by ATP was larger at lower than at higher concentration of the respective cosubstrate. These results fully confirm the proposed random mechanism