Terbium ions and
terbium formycin triphosphate have been used to investigate the interactions between the
cation and
nucleotide binding sites of the sarcoplasmic reticulum Ca2+-
ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to
magnesium binding sites, located near a
tryptophan residue of the
protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the
calcium transport sites, their occupancy by
terbium induces the E1 to E2 conformational change of the Ca2+-
ATPase; a third class of sites is revealed by following the fluorescence transfer from
formycin triphosphate (FTP) to
terbium, evidencing that
terbium ions can also bind into the
nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that
Tb-FTP binding to the
enzyme nucleotide site is associated with an important
dehydration of the
terbium ions associated with FTP. Two
terbium ions, at least, bind to the Ca2+-
ATPase in the close vicinity of FTP when this
nucleotide is bound to the
ATPase nucleotide site. Addition of
calcium quenches the fluorescence signal of the
terbium-FTP complex bound to the
enzyme.
Calcium concentration dependence shows that this effect is associated with the replacement of
terbium by
calcium in the transport sites, inducing the E2----E1 transconformation when
calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the
nucleotide site. Another interpretation is that the high-affinity
calcium sites are located very close to the
Tb-FTP complex bound to the
nucleotide site.