title: Binding of TNP-ATP and TNP-ADP to the non-catalytic sites of Escherichia coli F1-ATPase.
abstract: Using site-directed-tryptophan fluorescence, parameters for equilibrium binding of (Mg)TNP-ATP and (Mg)TNP-ADP to non-catalytic sites of Escherichia coli F1-ATPase were determined. All three non-catalytic sites showed the same affinity for MgTNP-ATP (Kd = 0.2 microM) or MgTNP-ADP (Kd = 6.5 microM) whereas even at concentrations of 100 microM no binding of uncomplexed TNP-ATP or TNP-ADP was observed. The results demonstrate that the three non-catalytic sites bind TNP-nucleotides non-cooperatively, and emphasize the importance of Mg2+ for non-catalytic-site nucleotide binding. Parameters for binding of (Mg)TNP-ADP to the three catalytic sites were also determined, and showed marked cooperativity. This work completes the set of thermodynamic parameters for equilibrium binding of (Mg)TNP-ATP and (Mg)TNP-ADP to all six nucleotide sites of F1, providing essential information to fully exploit the potential of these nucleotide analogs in studies of F1-ATPase.
authors.affiliation: University of Rochester Medical Center, Department of Biochemistry and Biophysics, NY 14642, USA.
authors.forename: J
authors.initials: J
authors.lastname: Weber
authors.forename: A E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D008274:Magnesium / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D005456:Fluorescent Dyes; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium