Characterization of nucleotide binding sites on chloroplast coupling factor 1.
A study of the equilibrium binding of ADP, 1,N6-ethenoadenosine diphosphate, adenylyl imidodiphosphate, and 1,N6-ethenoadenylyl imidodiphosphate to solubilized spinach chloroplast coupling factor 1 (CF1) has been carried out. All four nucleotides were found to bind to two apparently identical "tight" sites, with characteristic dissociation contants generally less than 10 muM. The binding to these "tight" sites is similar in the presence of Mg2+ and Ca2+, is stronger in 0.1 M NaC1 than in 20 mM Tris-C1, and is only slightly altered by heat activation. The slow rate of association of ADP and 1,N6-ethenoadenosine diphosphate at these sites rules out the possibility that they are catalytic sites for ATPase activity on the solubilized enzyme. A third tight site for adenylyl imidodiphosphate was found on the heat-activated enzyme. The dissociation constant for this interaction (7.6 muM) is similar to the adenylyl imidodiphosphate competitive inhibition constant for ATPase activity (4 muM). ADP, which inhibits ATPase activity but is not a strong competitive inhibitor, binds only weakly at a third site (dissociation constant greater than 70 muM). One mole of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted per mole of CF1 prevents ADP and adenylyl imidodiphosphate binding at the "catalytic" site and abolishes the ATPase activity. A model is proposed in which the "tight" nucleotide binding sites act as allosteric conformational switches for the ATPase activity of solubilizedCF1.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives; D000494:Allosteric Regulation; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology* / Q000378:metabolism; D005453:Fluorescence; D006358:Hot Temperature; D008433:Mathematics; D008956:Models, Chemical; D009574:Nitro Compounds / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism*; D010069:Oxadiazoles / Q000494:pharmacology; D010785:Photophosphorylation; D013329:Structure-Activity Relationship
D009574:Nitro Compounds; D009711:Nucleotides; D010069:Oxadiazoles; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate