The native mitochondrial F1-inhibitor protein complex carries out uni- and multisite ATP hydrolysis.
The rate of ATP hydrolysis under multi- and unisite conditions was determined in the native F1-inhibitor protein complex of bovine heart mitochondria (Adolfsen, R., MacClung, J.A., and Moudrianakis, E.N. (1975) Biochemistry 14, 1727-1735). Aurovertin was used to distinguish between hydrolytic activity catalyzed by the F1-ATPase or the F1-inhibitor protein (F1.I) complex. We found that incubation of aurovertin with the F1.I complex, prior to the addition of substrate, results in a stimulation of the hydrolytic activity from 1 to 8-10 mumol min-1 mg-1. The addition of aurovertin to a F1.I complex simultaneously with ATP results in a 30% inhibition with respect to the untreated F1.I. In contrast, if the F1.I complex is activated up to a hydrolytic activity of 80 mumol min-1 mg-1, aurovertin inhibits the enzyme in a manner similar to that described for F1-ATPase devoid of the inhibitor protein. The native F1.I complex catalyzes the hydrolysis of ATP under conditions for single catalytic site, liberating 0.16-0.18 mol of Pi/mol of enzyme. Preincubation with aurovertin before the addition of substrate had no effect under these conditions. On the other hand, if the F1.I ATPase was allowed to hydrolyze ATP at a single catalytic site, catalysis was inhibited by 98% by aurovertin. In F1-ATPase, the hydrolysis of [gamma-32P]ATP bound to the first catalytic site is promoted by the addition of excess ATP, in the presence or absence of aurovertin. Under conditions for single site catalysis, hydrolysis of [gamma-32P]ATP in the F1.I complex was not promoted by excess ATP. We conclude that the endogenous inhibitor protein regulates catalysis by promoting the entrapment of adenine nucleotides at the high affinity catalytic site, thus hindering cooperative ATP hydrolysis.
Vázquez-Laslop
N
N
Departamento de Bioenergética, Universidad Nacional Autónoma de México, D.F., México.
Dreyfus
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001313:Aurovertins / Q000494:pharmacology; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology; D011506:Proteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000097795:ATPase Inhibitory Protein
D000255:Adenosine Triphosphate; D001313:Aurovertins; D046911:Macromolecular Substances; D011506:Proteins; D006180:Proton-Translocating ATPases; D000097795:ATPase Inhibitory Protein