Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP.
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.
Girardet
J L
JL
Laboratoire de Biophysique Moléculaire et Cellulaire (Unité Associée 520 du Centre National de la Recherche Scientifique), France.
Dupont
Y
Y
Lacapere
J J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005573:Formycins / Q000378:metabolism*; D007700:Kinetics; D008962:Models, Theoretical; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D012265:Ribonucleotides / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D005573:Formycins; D012265:Ribonucleotides; D013725:Terbium; C051617:formycin triphosphate-terbium complex; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium