Effect of covalent binding of a derivative of 2',3'-O-(2,4,6-trinitrophenyl)-ADP to the tight binding site of CF1 on the enzyme activity.

Irradiation of isolated chloroplast thylakoids with TNP-ADP results in non-covalent binding and covalent incorporation of a reaction product of TNP-ADP formed by photosynthetic reduction into the so-called "tight" nucleotide binding site of CF1 [Ponse et al. (1992) Z. Naturforsch. 47c, 264-274]. CF1 extracted from thus-loaded thylakoid membranes yielded maximal incorporation of 1 mol/mol of CF1. Almost half had the covalent bond with CF1. In experiments with TNP-[14C]ADP, radioactivity was detected almost equivalently on alpha and beta subunits, suggesting that the binding site may be at the interface between alpha and beta subunits. Enzyme activities of the thylakoid membrane-bound enzyme after covalent labeling were measured. Inhibition, ranging from 20 to 25%, was less than expected from the percentage of CF1 molecules labeled (40-50%). It is suggested that only half of the labeled enzymes, probably those with the nucleotide analog linked to the beta subunit, are inactive.