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Studies of nucleotide binding to the catalytic sites of Escherichia coli βY331W-F1-ATPase using fluorescence quenching

Most studies of nucleotide binding to catalytic sites of Escherichia coli βY331W-F1-ATPase by the quenching of the βY331W fluorescence have been conducted in the presence of ≈20 mM sulfate. We find that, in the absence of sulfate, the nucleotide concentration dependence of fluorescence quenching induced by ADP, ATP, and MgADP is biphasic, revealing two classes of binding sites, each contributing about equally to the overall extent of quenching. For the high-affinity catalytic site, the Kd values for MgADP, ADP, and ATP equal 10, 43, and 185 nM, respectively. For the second class of sites, the Kd values for these ligands are ≈1,000× larger at 8.1, 37, and 200 μM, respectively. The presence of sulfate or phosphate during assay results in a marked increase in the apparent Kd values for the high-affinity catalytic site. The results show, contrary to earlier reports, that Mg2+ is not required for expression of different affinities for a nucleotide by the three catalytic sites. In addition, they demonstrate that the fluorescence of the introduced tryptophans is nearly completely quenched when only two sites bind nucleotide. Binding of ADP to the third site with a Kd near mM gives little fluorescence change. Many previous results of fluorescence quenching of introduced tryptophans appear to require reinterpretation. Our findings support a bi-site catalytic mechanism for F1-ATPase.