Characterization of nucleotide binding sites on membrane-bound chloroplast ATPase by modification with pyridoxal 5′-phosphate

Abstract

Treatment of chloroplast thylakoids with pyridoxal 5′-phosphate (PLP)in the light or in the presence of Mg 2+ causes inhibition of photophosphorylation which is partially competitive to ADP and to inorganic phosphate (P i ), suggesting that PLP may modify essential lysine residues in the catalytic nucleotide binding site of the thylakoid H + -ATPase. Treatment of thylakoids with PLP and NaB 3 H 4 results in incorporation of about 4 mol [ 3 H]PLP/mol CF 1 almost equally distributed between α- and β-subunits. ADP plus P i , prevents incorporation of one PLP per three α-subunits and one per three β-subunits, but causes almost full protection against PLP inactivation, suggesting that PLP modification of only one α- and one β-subunit is sufficient for inactivation of the enzyme. As PLP modification of the ATPase is largely excluded in the absence of Mg 2+ , modification of the active site may require ionic fixation of PLP with the help of the phosphate side-chain via Mg 2+ , similar to the interaction of P β and P γ of ATP with the protein in order to facilitate the covalent attack to the vicinal lysine residue.