Identification of an exchangeable non-catalytic 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 Mg(2+)-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 Vmax. With 8-azido-ADP two exchangeable non-catalytic sites could be 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 Km 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.
Edel
C M
CM
E.C. Slater Institute, University of Amsterdam, The Netherlands.
Hartog
A F
AF
Berden
J A
JA
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002384:Catalysis; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; C021622:8-azidoadenosine diphosphate