Inactivation of chloroplast H+‐ATPase by modification of Lysβ359, Lysα176 and Lysα266

Treatment of isolated, latent chloroplast ATPase with pyridoxal-5-phosphate (pyridoxal-P) in presence of Mg2+ causes inhibition of dithiothreitol-activated plus heat-activated ATP hydrolysis. The amount of [3H]pyridoxal-P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 ± 1 pyridoxal-P/ enzyme, almost equally distributed between the α- and β-subunits. Inactivation, however, is complete after binding of 1.5–2 pyridoxal-P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H-labeled pyridoxal-P into β- and α-subunits. Phosphate prevents labeling of the α-subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the α- and β-subunits. Cleavage of the pyridoxal-P-labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lysβ359, Lysα176 and Lysα266, which are closely related to proposed nucleotide-binding regions of the α- and β-subunits.