ALPHA-ASPARTATE-261 IS A KEY RESIDUE IN NONCATALYTIC SITES OF ESCHERICHIA-COLI F1-ATPASE

X-ray structure analysis of the noncatalytic sites of F-1-ATPase revea led that residue alpha-Asp(261) lies close to the Mg of bound Mg-5'-ad enylyl-beta,gamma imidodiphosphate. Here, the mutation alpha D261N was generated in Escherichia coil and combined with the alpha R365W mutat ion, allowing nucleotide binding at F-1 noncatalytic sites to be speci fically monitored by tryptophan fluorescence spectroscopy. Purified al pha D261N/alpha R365W F-1-ATPase showed catalytic activity similar to wild-type. An important feature was that, without any resort to nucleo tide depletion procedures, the noncatalytic sites in purified native e nzyme were already empty. Binding studies with MgATP, MgADP, and the c orresponding free nucleotides led to the following conclusions. Residu e alpha-Asp(261) interacts with the Mg of Mg-nucleotide in noncatalyti c sites and provides a large component of the binding energy (similar to 3 kcal/mol). It is the primary determinant of the preference of non catalytic sites for Mg-nucleotide. The natural ligands at these sites in wild-type enzyme are the Mg-nucleotides and free nucleotides bind p oorly. Under conditions where noncatalytic sites were empty, alpha D26 1N/alpha R365W F-1 showed significant hydrolysis of MgATP. This establ ishes unequivocally that occupancy of noncatalytic sites by nucleotide is not required for catalysis.