Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid
residue side chains in the catalytic site of Escherichia coli FI-ATPa
se was investigated. From the X-ray structure of the mitochondrial enz
yme [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 b
eta 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 interveni
ng 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 inv
olved in Mg2+ coordination. whereas beta Glu-181 is not. A derived str
uctural model for the octahedral coordination around the Mg2+ ion is p
resented. The results indicate that the ADP-containing site in the X-r
ay structure is the catalytic site of highest affinity. Correct Mg2+ c
oordination is required for catalytic activity at physiological rates.
Mg2+-coordinating residues led to complete loss of Mg2+-dependent nuc
leotide binding cooperativity of the catalytic sites.