SLOW BINDING OF ATP TO NONCATALYTIC NUCLEOTIDE BINDING-SITES WHICH ACCELERATES CATALYSIS IS RESPONSIBLE FOR APPARENT NEGATIVE COOPERATIVITY EXHIBITED BY THE BOVINE MITOCHONDRIAL F1-ATPASE

The bovine heart mitochondrial F1-ATPase depleted of nucleotides (nd-MF1) hydrolyzes 50 muM ATP in three kinetic phases at 30-degrees-C. An initial ''burst'' rapidly transforms into an intermediate, slower rate, which slowly accelerates to the final, steady-state rate. The intermediate phase disappears progressively as the concentration of ATP in the assay medium is increased and is absent at 2 mM. Activation in the intermediate phase is lost when nd-MF1 is inactivated by 5'-p-fluorosulfonylbenzoyladenosine, which modifies three noncatalytic sites. Correlation of [H-3]ATP binding to nd-MF1, after treatment either with 50 muM Mg[H-3]ATP plus a regenerating system or 10 mM free [H-3]ATP, with stimulation of the intermediate phase suggests that this phase is abolished when at least two noncatalytic sites are filled with ATP. Prior incubation of nd-MF1 with MgPP(i) stimulates hydrolysis of 30 muM to 2 mM ATP and abolishes the intermediate phase. Following incubation with Mg[P-32]PP(i), 3.3 mol of [P-32]PP(i)/mol of enzyme are bound, 1 and 0.5 mol of which are released by cold chases with MgATP and MgITP, respectively. Since the cold chases diminish activation only slightly, the stimulatory effect is not caused by PP(i) binding to catalytic sites. A Lineweaver-Burk plot of initial rates of the intermediate phase for hydrolysis of 30 muM to 2 mm ATP by nd-MF1 is biphasic, extrapolating to apparent K(m) values of 120 and 440 muM. The latter value is the same as the apparent K(d) determined from dependence of the rate of activation of the intermediate phase on ATP concentration in the assay medium. After prior incubation of nd-MF1 with MgPP(i) or free ATP, Lineweaver-Burk plots are linear with the highest K(m) disappearing. Thus, this K(m) reflects rate acceleration when ATP binds to noncatalytic sites. From these results it is concluded that slow binding of ATP to noncatalytic sites during hydrolysis of low concentrations of substrate, which accelerates catalysis, is responsible for apparent negative cooperativity exhibited by MF1.