NMR studies identify four intermediate states of ATPase and the ion transport cycle of sarcoplasmic reticulum Ca2+-ATPase.
Water proton nuclear relaxation measurements are used to detect and characterize four distinct intermediate states for Gd3+ bound to Ca2+ sites of sarcoplasmic reticulum Ca2+-ATPase in complexes with ATP analogues. In the absence of nucleotides, Gd3+ binds to two occluded Ca2+ transport sites on Ca2+-ATPase which have a low accessibility to solvent water. In the presence of the nonhydrolyzable ATP analogue, Co(NH3)4AMPPCP, a new state for bound Gd3+ (still occluded and with fewer waters of hydration) is observed. In the presence of Co(NH3)4ATP or ATP, two additional states for bound Gd3+ are detected in the NMR studies. The first of these probably represents an intermediate state for bound Gd3+ during ATP hydrolysis. The latter is the most occluded Gd3+ site yet observed in these studies and is probably analogous to the highly occluded E1-P state observed with CrATP [(1987) Biochim. Biophys. Acta 898, 313-322].
Klemens
M R
MR
Department of Chemistry, University of Virginia, Charlottesville 22901.
Grisham
C M
CM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005682:Gadolinium / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000255:Adenosine Triphosphate; D005682:Gadolinium; D000252:Calcium-Transporting ATPases; D002118:Calcium