Influence of divalent cations on nucleotide exchange and ATPase activity of chloroplast coupling factor 1

The ATPase activity of the catalytic part of ATP synthases is inhibited by free Mg2+, even though MgATP is the substrate. Here we show that the inhibi tion of the MgATPase activity of chloroplast coupling factor 1 deficient in its epsilon subunit (CF1-epsilon) by Mg2+ is complex. The hydrolysis of Mg ATP by CF1-epsilon that contains tightly bound ADP, but no bound Mg2+, is i nitially rapid and decreases within about I min to a steady-state rate. The bound MgADP content of CF1-epsilon was varied. The initial fast phase of M gATP hydrolysis is eliminated when the molar ratio of MgADP to CF1-epsilon approaches 2. Loosely bound Mg2+ also affects the initial kinetics of the e nzyme that contains bound MgADP. At molar ratios of bound MgADP to enzyme i n excess of 1, the initial ATPase activity was low and reached the steady s tate after about 30 s. Free Mg2+ in the assay mix also inhibited steady-sta te ATP hydrolysis by all forms of the enzyme. The results are consistent wi th a model in which two Mg2+ bind cooperatively, probably to the dissociabl e nucleotide-binding sites on CF1-epsilon Thus, four different nucleotide-b inding sites may be involved in the inhibition of the MgATPase activity of CF1-epsilon. Three of these sites are potentially catalytic, and the fourth may be regulatory. The exchange of bound trinitrophenyl-ADP induced by the addition of MgATP or CaATP was found to be fast enough for the sire to be involved in catalysis.