Binding of TNP‐ATP and TNP‐ADP to the non‐catalytic sites of Escherichia coli F1‐ATPase

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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 (K d=0.2 μM) or MgTNP‐ADP (K d=6.5 μM) whereas even at concentrations of 100 μM 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.