Binding of TNP-ATP and TNP-ADP to the non-catalytic sites of Escherichia coli F-1-ATPase

Using site-directed-tryptophan fluorescence, parameters for equilibrium binding of (Mg)TNP-ATP and (Mg)TNP-ADP to non-catalytic sites of Escherichia coli F-1-ATPase were determined. All three non-catalytic sites showed the same affinity for MgTNP-ATP (K-d = 0.2 mu M) or MgTNP-ADP (K-d = 6.5 mu M) whereas even at concentrations of 100 mu M 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-she 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 F-1, providing essential information to fully exploit the potential of these nucleotide analogs in studies of F-1-ATPase. (C) 1997 Federation of European Biochemical Societies.