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 IT P. After incubation of nd-MF1 with free ADP, inhibition develops hyste retically 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 bi nding of [C-14]ADP, in the presence or absence of Mg2+, with the exten t of hysteretic inhibition induced suggests that maximal inhibition oc curs when at least two noncatalytic sites are filled with ADP. Hystere tic 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-P-32]ADP, in the presence or absence of Mg2+, increases th e extent of induced inhibition which correlates with increasing deriva tization of tyrosine beta368 following irradiation of loaded enzyme. T his demonstrates that binding of ADP to noncatalytic sites is, in part , responsible for induction of hysteretic inhibition. After inducing i nhibition by prior incubation with ADP, the steady-state kinetic behav ior 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 un inhibited enzyme. The composite results suggest that prior saturation of noncatalytic sites of nd-MF1 with ADP prevents activation of the en zyme by blocking the binding of ATP to these sites which is necessary to promote dissociation of inhibitory MgADP from a catalytic site.