Photoaffinity labeling of the TF₁-ATPase from the thermophilic bacterium PS3 with 3′-O-(4-benzoyl)benzoyl ADP

(1) 3′-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF₁). (2) As with the CF₁-ATPase (Bar-Zvi, D. and Shavit, N. (1984) Biochim. Biophys. Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF₁-ATPase. BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CF₁-ATPase, it has no effect on the V_max. In the absence of Mg²⁺ 1 mol BzADP binds noncovalently to TF₁, while with Mg²⁺ 3 mol are bound. (3) Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF₁ and correlates with the inactivation of the ATPase. Complete inactivation of the TF₁-ATPase occurs after covalent binding of 2 mol BzADP / mol TF₁. Photoinactivation of TF₁ by BzADP is prevented if excess of either ADP or ATP is present during irradiation. (4) Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bz[³H]ADP-labeled TF₁-ATPase shows that all the radioactivity is incorporated into the β subunit.