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 microM 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 [3H]ATP binding to nd-MF1, after treatment either with 50 microM Mg[3H]ATP plus a regenerating system or 10 mM free [3H]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 MgPPi stimulates hydrolysis of 30 microM to 2 mM ATP and abolishes the intermediate phase. Following incubation with Mg[32P]PPi, 3.3 mol of [32P]PPi/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 PPi binding to catalytic sites. A Lineweaver-Burk plot of initial rates of the intermediate phase for hydrolysis of 30 microM to 2 mM ATP by nd-MF1 is biphasic, extrapolating to apparent Km values of 120 and 440 microM. The latter value is the same as the apparent Kd 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 MgPPi or free ATP, Lineweaver-Burk plots are linear with the highest Km disappearing. Thus, this Km 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.