The use of 8-azido-ATP and 8-azido-ADP as photoaffinity labels of the ATP synthase in submitochondrial particles: evidence for a mechanism of ATP hydrolysis involving two independent catalytic sites?
8-Azido-ATP is a substrate for the ATP synthase in submitochondrial particles with a Vmax equal to 6% of the Vmax with ATP. The Km values for 8-azido-ATP are similar to those for ATP. ATP synthase in submitochondrial particles can bind maximally 2 mol 8-N-ATP or 8-N-ADP per mole and the inhibition of ATP hydrolysis by covalently bound N-ATP or N-ADP is proportional to the saturation of the enzyme with inhibitor, similar to the results obtained with isolated F1. Both 8-N-ATP and 8-N-ADP are bound mainly to the beta subunits and at all levels of saturation the distribution of the label is 77% to the beta and 23% to the alpha subunits. It is proposed that the binding of 8-azido-AXP itself is mainly to the beta subunit, but that part of the nitreno radicals formed during excitation with light reacts with an amino acid of the alpha subunit, due to the location of the binding site at an interface between a beta and an alpha subunit. Partial saturation with 8-N-ATP, under conditions that the concentration of 8-azido-ATP during the incubation is intermediate between the low and high Km values, does not abolish the apparent negative cooperativity of ATP hydrolysis. It is concluded that this apparent cooperativity is not due to the presence of two different catalytic sites, nor to a cooperativity between the two catalytic sites, but to interaction between the catalytic sites and regulatory sites.
Sloothaak
J B
JB
Berden
J A
JA
Herweijer
M A
MA
Kemp
A
A
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D004789:Enzyme Activation / Q000528:radiation effects; D005609:Free Radicals; D006868:Hydrolysis; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D011485:Protein Binding / Q000528:radiation effects; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D005609:Free Radicals; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; C021622:8-azidoadenosine diphosphate