Mg2+ Coordination in Catalytic Sites of F1-ATPase

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Abstract
Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli F1-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. (1994) Nature 370, 621−628], it may be inferred that the hydroxyl of βThr-156 is a direct ligand of Mg2+, whereas the carboxyls of βGlu-181, βGlu-185, and βAsp-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 βThr-156, βGlu-185, and βAsp-242 are all involved in Mg2+ coordination, whereas βGlu-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. Elimination of any one of the Mg2+-coordinating residues led to complete loss of Mg2+-dependent nucleotide binding cooperativity of the catalytic sites.