Mechanistic basis for differential inhibition of the F1Fo-ATPase by aurovertin.
The mitochondrial F(1)F(o)-ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F(1)F(o)-ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the beta subunits in the F(1) domain and inhibits F(1)F(o)-ATPase-catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F(1)F(o)-ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K(i) values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F(1)F(o)-ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single-molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F(1)F(o)-ATPase catalyzes ATP synthesis and hydrolysis.
Johnson
Kathryn M
KM
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Swenson
Lara
L
Opipari
Anthony W
AW
Reuter
Rolf
R
Zarrabi
Nawid
N
Fierke
Carol A
CA
Börsch
Michael
M
Glick
Gary D
GD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001313:Aurovertins / Q000494:pharmacology*; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology*
D000900:Anti-Bacterial Agents; D001313:Aurovertins; D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases