Interaction of nucleotide-depleted F 1-ATPase with ADP : Characterization of a high-affinity binding site responsible for ADP-induced inhibition

Abstract

The ADP-induced inhibition of nucleotide-depleted F1- ATPase (ndF1-ATPase) from bovine heart mitochondria has been shownto be the result of ADP binding at one site with a dissociation constant of 4–5 nM. ADP binding at this site is at least biphasic: the initially formed inactive complex (ADP · ndF1) dissociating with the rate of about 0.01 s−1 is transformed with a half-time of 2–3 min to another state (ADP · ndF1*) with the ADP-release rate of about 4×10 −4 s−1. ATP and ADP were found to accelerate the dissociation of ADP from the inhibitory site; half-maximal effects were exerted by 170 μM ATP and 120 μM ADP. The ATP-dependent reactivation of the inactive complex of ndF1-ATPase and ADP is suggested to result from the ATP-induced shift of the equilibrium between the ADP · ndF1 and ADP · ndF 1* complexes to the former one. The nature of the inhibitory ADP-binding site is finally discussed, and it is concluded that this ADP-binding site is clearly distinct from the catalytic sitesparticipating in the normal turnover of the enzyme.