Interaction of high-affinity nucleotide binding sites in mitochondrial ATP synthesis and hydrolysis.
The present study contributes to the problem of the dynamic structure of mitochondrial F1-ATPase and the functional interrelation of so-called tight nucleotide binding sites. Nucleotide analogs are used as a tool to differentiate two distinct functional states of the membrane-bound enzyme, proposed to reflect corresponding conformational states; they reveal F1-ATPase as a "dual-state" enzyme: ATP-synthetase, and ATP-hydrolase. The analogs used are 3'-naphthoyl esters of AD(T)P, and 2'(3')-O-trinitrophenyl ethers of AD(T)P. Both types of analogs act inversely to each other with respect to their relative effects on oxidative phosphorylation and on ATPase in submitochondrial vesicles. The respective ratios of Ki versus both processes are 250/1 compared to 1/170. It is also shown that in the presence of the inhibitory 3'-esters oxidative phosphorylation deviates from linear kinetics and that these inhibitors induce a lag time of oxidative phosphorylation depending on the initial pattern of nucleotides available to energized submitochondrial vesicles. The duration of the lag time coincides with the time course of displacement of the analog from a tight binding site. The conclusions of the study are: (a) the catalytic sites of F1-ATP-synthetase are not operating independently from each other; they rather interact in a cooperative manner; (b) F1-ATPase as a "dual-state" enzyme exhibits highly selective responses to tight binding of nucleotides or analogs in its "energized" (membrane-bound) state versus its "nonenergized" state, respectively.
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008930:Mitochondria, Liver / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D006180:Proton-Translocating ATPases / Q000378:metabolism
D009711:Nucleotides; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases