α-Aspartate 261 is a key residue in noncatalytic sites of Escherichia coli F₁-ATPase

X-ray structure analysis of the noncatalytic sites of F₁-ATPase revealed that residue α-Asp²⁶¹ lies close to the Mg of bound Mg-5'-adenylyl-β- γ-imidodiphosphate. Here, the mutation αD261N was generated in Escherichia coli and combined with the αR365W mutation, allowing nucleotide binding at F₁ noncatalytic sites to be specifically monitored by tryptophan fluorescence spectroscopy. Purified αD261N/αR365W F₁-ATPase showed catalytic activity similar to wild-type. An important feature was that, without any resort to nucleotide-depletion procedures, the noncatalytic sites in purified native enzyme were already empty. Binding studies with MgATP, MgADP, and the corresponding free nucleotides led to the following conclusions. Residue α-Asp²⁶¹ interacts with the Mg of Mg-nucleotide in noncatalytic sites and provides a largo component of the binding energy (~3 kcal/mol). It is the primary determinant of the preference of noncatalytic sites for Mg-nucleotide. The natural ligands at these sites in wild-type enzyme are the Mg-nucleotides and free nucleotides bind poorly. Under conditions where noncatalytic sites were empty, αD261N/αR365W F₁ showed significant hydrolysis of MgATP. This establishes unequivocally that occupancy of noncatalytic sites by nucleotide is not required for catalysis.