NUCLEOTIDE-BINDING TO CHLOROPLAST F1-ATPASE (CF1) CANNOT BE EXPLAINEDBY A 2-SITE MODEL

The kinetics of nucleotide binding to the chloroplast coupling factor, CF1, have been analysed by stopped-flow measurements using the fluore scent trinitrophenyl analog TNP-ADP. Experimental determined time cour ses were compared with several different theoretical time courses. The following observations were obtained: (a) in stopped-flow experiments the half time of the reaction was at highest with a ligand-to-enzyme ratio of 2:1. The half time of the binding reaction decreased, if the ratio was lower than 2:1. (b) This characteristic cannot be explained by a bimolecular reaction mechanism of several independent (classes of ) binding sites. (c) Theoretical lime courses according to a ''two-sit e'' model with the inclusion of a conformational change of each site a fter binding of ligand showed the observed characteristic (see (a)). ( d) However, this ''two-site'' model including a conformational change of each site after binding of ligand failed to simulate the measured t ime courses of nucleotide binding.