title: Alteration of the nucleotide-binding site asymmetry of chloroplast coupling factor 1 by catalysis.
abstract: Fluorescence resonance energy transfer was used to show that ATP hydrolysis induces a change in the properties of two nucleotide-binding sites in isolated chloroplast coupling factor 1 (CF1). The fluorescence donor was Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide- 3,6-disulfonate), covalently bound to a unique site on the alpha subunit between nucleotide-binding sites 2 and 3. The fluorescence acceptor was the ATP analog 2'(3')-trinitrophenyladenosine 5'-triphosphate (TNP-ATP), incorporated specifically into nucleotide-binding site 1. Energy transfer from Lucifer Yellow to TNP-ATP in site 1 was greater if catalysis occurred before TNP-ATP was incorporated than if no catalysis occurred, indicating that one of the nucleotide-binding sites near the Lucifer Yellow had changed its properties to those of site 1 as a result of catalysis. The amount of energy transfer increased with the degree of substrate excess during catalysis, as expected if catalysis were required for the new site 1 location. ADP, which binds to CF1, but is not a substrate for hydrolysis, caused little energy transfer. Titration of site 3 with TNP-ATP showed greater energy transfer from Lucifer Yellow when catalysis had not occurred, indicating that sites 1 and 3 switched properties as a result of catalysis. The amount of energy transfer declined exponentially with time between removal of substrate and addition of TNP-ATP to site 1, with a half-time of 1.5-2 h at room temperature. This result suggests that the change that results in switching of nucleotide-binding sites 1 and 3 relaxes in the absence of substrate. Our results show that the asymmetry of the nucleotide-binding sites of CF1 is not a permanent feature of the molecule.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D007546:Isoquinolines; D000255:Adenosine Triphosphate; C017475:lucifer yellow; D006180:Proton-Translocating ATPases