IDENTIFICATION OF AN EXCHANGEABLE NONCATALYTIC SITE ON MITOCHONDRIAL F1-ATPASE WHICH IS INVOLVED IN THE NEGATIVE COOPERATIVITY OF ATP HYDROLYSIS

Labeling of mitochondrial F1-ATPase with 8-azido-ATP or 8-azido-ADP under turnover conditions with Mg2+-ATP resulted in the identification of one exchangeable non-catalytic site whose occupation with a ligand does not influence the ATPase activity of F1 when measured at V(max). With 8-azido-ADP two exchangeable non-catalytic sites could bc labeled, but at one of them the bound ligand exchanges, at least partly, during the illumination under turnover conditions. After labeling an exchangeable non-catalytic site under turnover conditions with 8-azido-ATP or with 8-azido-ADP, F1-ATPase kept the ability to bind NAP3-2N3ADP at the slowly exchangeable noncatalytic site, thereby inhibiting the ATPase activity by 45%, as recently described (Edel et al. (1992) Biochim. Biophys. Acta 1101, 329-338). Covalent modification of the low-affinity non-catalytic site with 8-nitreno-AT(D)P increased the K(m) of ATP and abolished the negative cooperativity of ATP hydrolysis. This site can therefore be marked as a regulatory site, whose occupation with a nucleotide decreases the affinity of the catalytic sites for ATP.