Mg2+ coordination in catalytic sites of F1-ATPase

Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli FI-ATPase was investigated. From the X-ray structure of the mitochondrial enzyme [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1944) Nature 370, 621-628], it may be inferred that the hydroxyl of beta Thr-156 is a direct ligand of Mg2+, whereas the carboxyls of beta Glu-181, beta Glu-185, and beta Asp-242 might contribute via intervening water molecules Elimination of each respective functional group by site-directed mutagenesis, followed by determination of Mg-nucleotide and uncomplexed nucleotide binding affinities using a tryptophan probe, showed that beta Thr-156. beta Glu-185, and beta Asp-242 are all involved in Mg2+ coordination. whereas beta Glu-181 is not. A derived structural model for the octahedral coordination around the Mg2+ ion is presented. The results indicate that the ADP-containing site in the X-ray structure is the catalytic site of highest affinity. Correct Mg2+ coordination is required for catalytic activity at physiological rates. Mg2+-coordinating residues led to complete loss of Mg2+-dependent nucleotide binding cooperativity of the catalytic sites.