Abstract
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.
Highlights
Chemical modification studies with 2-azido-ATP have provided evidence that Tyr-345 of the 0-subunit is located at catalytic sites of the beef heart enzyme, whereas Tyr-368 is located at noncatalytic sites [6].Selective modification of the catalytic or the noncatalytic site haaslso been achieved using two other nucleotide analogues, FSBI’ and FSBA
That noncatalytic sites areinvolved in control of catalysis only slightly, the stimulatory effect is not caused by has been suggested on the basis of several experimental cri
Steady-state kinetic analyses of initial ratesof the intermediate phase for hydrolysiosf F1-ATPases from different sources have revealed apparent of 30 WM to 2 mM ATP by nd-MF1 is biphasic, extrap- negative cooperativity whenATP hydrolysis is measured over olating to apparenKt, values of 120 and 440 p ~ T.he a wide range of substrate concentration
Summary
A n Activation PhaseI s Exhibited When nd-MFH1 ydrolyzes Low,but Not High Concentrationsof ATP-Hydrolysis of 50 P M ATP by nd-MF1proceeds inthree distinct phases sahsown in Fig. 1 (trace a ). Prior incubationof nd-MF1 with mM PPIin the presence of Mg2+ abolishes the intermediate phase observed during hydrolysis of 50 PM ATP, and stimulates ATPase activity during assay a t low and high ATP concentrations as shown by comparison of traces c with traces ain Fig. 2 Recordings for hydrolysis of 50 PM ATP by samples of ndMFl undergoing inactivation by FSBA become more linear as noncatalyticsitesare increasingly modified This suggests that the progressive increase in activity observed during the FIG. Correlation of the disap- by addition of ATP andMgC1, to final concentrationsof 2 mM each,
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