THE KINETICS OF PHOTOPHOSPHORYLATION AT CLAMPED DELTA-PH INDICATE A RANDOM ORDER OF SUBSTRATE-BINDING

The rate of photophosphorylation of isolated chloroplast thylakoids wa s investigated at a clamped Delta pH of 2.5 (Delta Psi = 0). On variat ion of the concentration of ADP at different fixed concentrations of p hosphate, straight lines were obtained in double-reciprocal plots whic h intersected in one point below the x-axis. Upon variation of the pho sphate concentration at several fixed concentrations of ADP, similar k inetics were found. These results indicate a random order of binding o f the two substrates. Calculation of the dissociation constants and Mi chaelis constants from these two sets of data according to a bisubstra te rapid equilibrium random model yielded satisfactory agreement. The kinetic constants were found to be unchanged by thiol modulation or de modulation 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 respect ive cosubstrate. These results fully confirm the proposed random mecha nism.