Properties of the tight nucleotide binding site of chloroplast coupling factor CF1 deficient in delta- and epsilon-subunits

The effect of the pH of the medium on the kinetics of the interaction of [C-14]ATP and [C-14]ADP with CF1-delta epsilon at substoichiometric nucleotide/enzyme ratios was studied. Decreasing the pH from 8.0 to 6.0 results in a manyfold increase of the rate and extent of tight ATP binding, whereas ADP binding changes only slightly. The rate of hydrolysis of tightly bound ATP decreases; ATP dissociation becomes slower, while ADP dissociation slightly accelerates; the dissociation yields much more AMP. The AMP is shown to be produced by hydrolysis of ADP. Calculations show that acidification of the medium to pH 6.0 results in a 200-fold increase in tight nucleotide binding site affinity for ATP accompanied by a 3.5-fold decrease in its affinity for ADP. This suggests that the changes in site affinity and selectivity are controlled by protonation/deprotonation of specific acid-base groups located within the alpha-gamma subunits of CF1; protonation of these groups promotes stabilization of ATP binding while their deprotonation contributes to ADP binding.