Binding and hydrolysis of 2-azido-ATP and 8-azido-ATP by isolated mitochondrial F1: characterisation of high-affinity binding sites.
The kinetic parameters for the hydrolysis by F1 of the photoreactive nucleotide analogue 2-azido-ATP were determined (Vmax, 105 U/mg F1; Km, 250 microM, in the presence of 1.0 mM SO2-3). In the absence of an activating anion, a non-linear relationship in a Lineweaver-Burk plot was found for the hydrolysis of 2-azido-ATP. The 2-azido-analogues of ATP and ADP proved to be good photoaffinity labels causing notable inactivation of the F1-ATPase activity upon irradiation at 360 nm. This inhibition was also used to demonstrate high-affinity binding of these analogues to a catalytic binding site on the F1. High-affinity binding proved to be an Mg2+-requiring process, occurring with both 2-azido-ATP and 2-azido-ADP but hardly or not occurring with 8-azido-AT(D)P. Covalent binding of 2-nitreno-ATP upon irradiation of F1 containing tightly bound [beta-32P]2-azido-ATP results in a proportional inhibition of ATPase activity, extrapolating to 0.92 mol of covalently bound label per mol of F1 needed for the complete inactivation of the enzyme. When the F1 was irradiated in the presence of excess [beta-32P]2-azido-AT(D)P, 3-4 mol of label were bound when the enzyme was fully inactivated. In all cases, all or most of the radioactivity was found on the beta subunits.
van Dongen
M B
MB
de Geus
J P
JP
Korver
T
T
Hartog
A F
AF
Berden
J A
JA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism*; D010777:Photochemistry; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; C017040:8-azidoadenosine 5'-triphosphate; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases