Modification of membrane-bound F-1 by p-fluorosulfonylbenzoyl-5'-adenosine: Sites of binding and effect on activity

Bovine heart submitochondrial particles (smp) were incubated with p-fluorosulfonylbenzoyl-5'-adenosine (FSBA) in order to study the binding of this ligand and its effect on ATP synthesis and ATP hydrolysis in smp and to compare the results with those obtained with isolated F-1. The binding was measured with the C-14-labeled compound. ATP hydrolysis was in all cases as much inhibited as succinate-driven ATP synthesis and ITP hydrolysis was more inhibited than ATP hydrolysis. The binding experiments show that modification of three nucleotide binding sites results in nearly complete inhibition of ATPase activity. In the presence of pyrophosphate up to 6 mol [C-14]SBA/mol F-1 can be bound. FSBA preferentially modifies amino acids of the alpha-subunits but also beta-subunits are modified. It is concluded that modification of both subunits results in inhibition of activity. The results are very well comparable with the results obtained with isolated F-1, which indicates that our preparation of F-1 is a good model for F-1 in the intact system. Furthermore it is concluded that each alpha-subunit of F-1 in smp, just like in the isolated form, contains two pockets where adenosine moieties can bind, one located above the P-loop, modifying alpha-Tyr-244 and alpha-Tyr-300 and the other one located below the P-loop where also the adenosine moiety of AD(T)P binds, modifying beta-Tyr-368.