The Heterogeneous Interaction of Substoichiometric TNP-ATP and Fi-ATPase from Escherichia coli

The interactions of substoichiometric TNP-ATP and F,-ATPase from Escherichia coli (EF,) were examined and compared with those in the case of mitochondrial F,-ATPase (MF,) and F,-ATPase from thermophilic Bacillus PS3 (TF,). EF, hydrolyzed substoichio-metric TNP-ATP faster than TF, or MF,, although some 20 % of the TNP-ATP remained unhydrolyzed even in the presence of excess chase ATP. The affinity of the catalytic site of EF, for the product, TNP-ADP, was weaker than that of TF, or MF,, and the TNP-ADP was readily released upon addition of excess ATP. The amplitude of the difference absorption spectrum induced by binding of TNP-AT(D)P to EF, was smaller than that of MF, or TF, under similar experimental conditions. When an excess amount of TNP-ATP was added to EF, and the change of the difference spectrum was measured, the shape of the difference spectrum of the ATP-replaceable fraction was very similar to that in the case of binding of TNP-ATP to the isolated 0 subunit of TF,, indicating that the rapidly replaceable fraction of bound TNP-ATP was actually at the catalytic site and most of the non-replaceable portion was bound at noncatalytic sites. Weaker affinity of the catalytic site for TNP-ATP may account for the heterogeneous binding and hydrolysis under the conditions describe