Exploring the Adenine Nucleotide Binding Sites on Mitochondrial F1- ATPase with a New Photoaffinity Probe, 3’-0-(4-Benzoyl)benzoyl Adenosine 5’-Triphosphate*

3‘-0-(4-Benzoyl)benzoyl ATP (BzATP) was synthesized and used as a photoactivatable, covalently binding affinity probe to study site-specific adenine nucleotide binding to the ATPase of submitochondrial particles and the purified soluble F1-ATPase of rat liver mitochondria. In the absence of actinic light, BzATP was a good substrate for enzymic hydrolysis with both soluble and membrane-bound F1-ATPase. Photolysis of either the membrane-bound or soluble ATPase complex in the presence of BzATP resulted in the covalent incorporation of analog and a concomitant loss of enzyme activity. Yet, 4-benzoylbenzoic acid, the photoreactive functional group of the ATP analog, did not by itself inhibit enzyme activity after photolysis. Photoaffinity labeling with BzATP caused a rapid and significant decrease in V, (>70%) with no effect on the apparent K,,, of either submitochondrial particles or the soluble F1-ATPase complex. Inhibition of submitochondrial particle-ATPase activity by BzATP was found to be more efficient in the presence than in the absence of M&+. Resolution via polyacrylamide gel electrophoresis of the component subunit polypeptides of soluble F,-ATPase, photolabeled with [‘HIor [y-”P]BzATP, showed that all of the radioactivity was incorporated into only the a and p subunits of the enzyme. Radioactivity was found in both the a and ,6 subunits when [2,8-3H]BzATP was photolyzed in the presence of M&+, whereas only the /3 subunit was labeled in the absence of M S + . On the other hand, incorporation of label with [y-”P] BzATP always appeared exclusively in the p subunit whether or not Mg2+ was present. A mechanism of enzyme action for the Fl-ATPase is presented based on these and other recent data. The proposed mechanism suggests that a catalytic site resides on the p subunit. This site binds ATP-(M$’) and is closely associated, both topologically and hnctionally, with a specific ADP binding site situated on an immediately apposing a subunit.