Hysteretic inhibition of the bovine heart mitochondrial F1-ATPase is due to saturation of noncatalytic sites with ADP which blocks activation of the enzyme by ATP.

Prior incubation of the bovine heart mitochondrial F1-ATPase depleted of endogenous nucleotides (nd-MF1) with saturating ADP in the presence or absence of Mg2+ induces inhibition of hydrolysis of 2 mM ATP or ITP. After incubation of nd-MF1 with free ADP, inhibition develops hysteretically which is characterized by an uninhibited initial rate which decelerates to an inhibited, steady-state rate. When prior incubation of nd-MF1 is performed with ADP in the presence of Mg2+, the enzyme is partially inhibited when diluted into assay medium and more extensive inhibition develops hysteretically during turnover. Correlation of binding of [14C]ADP, in the presence or absence of Mg2+, with the extent of hysteretic inhibition induced suggests that maximal inhibition occurs when at least two noncatalytic sites are filled with ADP. Hysteretic inhibition is also induced by prior incubation of the enzyme with 2-N3-ADP. Prior incubation of nd-MF1 with increasing concentrations of 2-N3-[beta-32P]ADP, in the presence or absence of Mg2+, increases the extent of induced inhibition which correlates with increasing derivatization of tyrosine beta 368 following irradiation of loaded enzyme. This demonstrates that binding of ADP to noncatalytic sites is, in part, responsible for induction of hysteretic inhibition. After inducing inhibition by prior incubation with ADP, the steady-state kinetic behavior of nd-MF1 differs from that of uninhibited enzyme. Lineweaver-Burk plots of steady-state rates of inhibited enzyme as a function of ATP concentration are linear rather than biphasic which is observed for uninhibited enzyme. The composite results suggest that prior saturation of noncatalytic sites of nd-MF1 with ADP prevents activation of the enzyme by blocking the binding of ATP to these sites which is necessary to promote dissociation of inhibitory MgADP from a catalytic site.