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 mere 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 uncomplexcd TNP-ATP or TNP-ADP was observed. The results demonstrate that the three non-catalytic sites bind TNP-nucleotidcs 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 mere 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.
- Nucleotide binding site
- Oxidative phosphorylation