Adenine nucleotide binding sites on beef heart F1-ATPase. Asymmetry and subunit location.
Previously we have shown that beef heart mitochondrial F1 contains a total of six adenine nucleotide binding sites. Three "catalytic" sites exchange bound ligand rapidly during hydrolysis of MgATP, whereas three "noncatalytic" sites do not. The noncatalytic sites behave asymmetrically in that a single site releases bound ligand upon precipitation of F1 with ammonium sulfate. In the present study, we find this same site to be the only noncatalytic site that undergoes rapid exchange of bound ligand when F1 is incubated in the presence of EDTA at pH 8.0. Following 1000 catalytic turnovers/F1, the site retains the unique capacity for EDTA-induced exchange, indicating that the asymmetric determinants are permanent and that the three noncatalytic sites on soluble F1 do not pass through equivalent states during catalysis. Measurements of the rate of ligand binding at the unique noncatalytic site show that uncomplexed nucleotide binds preferentially. At pH 7.5, in the presence of Mg2+, the rate constant for ADP binding is 9 X 10(3) M-1 s-1 and for dissociation is 4 X 10(-4) s-1 to give a Kd = 50 nM. The rate of dissociation is 10 times faster in the presence of EDTA or during MgATP hydrolysis, and it increases rapidly at pH below 7. EDTA-induced exchange is inhibited by Mg2+, Mn2+, Co2+, and Zn2+ but not by Ca2+ and is unaffected by dicyclohexylcarbodiimide modification. The unique noncatalytic site binds 2-azido-ADP. Photolysis results in the labeling of the beta subunit. Photolabeling of a single high-affinity catalytic site under conditions for uni-site catalysis also results in the labeling of beta, but a different pattern of labeled peptides is obtained in proteolytic digests. The results demonstrate the presence of two different nucleotide binding domains on the beta subunit of mitochondrial F1.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D046911:Macromolecular Substances; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D006180:Proton-Translocating ATPases; D008274:Magnesium