Kinetics of Ca2+ binding to the SR Ca-ATPase in the E1 state.
The time-resolved kinetics of Ca2+ binding to the SR Ca-ATPase in the E1 state was investigated by Ca(2+)-concentration jump experiments. Ca2+ was released by an ultraviolet-light flash from caged calcium, and charge movements in the membrane domain of the ion pumps were detected by the fluorescent styryl dye 2BITC. The partial reaction (H3E1 <-->) E1 <--> CaE1 <--> Ca2E1 can be characterized by two time constants, tau1 and tau2, both of which are not significantly Ca(2+)-concentration-dependent and only weakly pH-dependent at pH < 7.5. Both time constants differ by a factor of approximately 50 (4.7 vs. 200 ms). The weak substrate-dependence indicates that the rate-limiting process is not related to Ca2+ migration through the access channel and ion binding to the binding sites but to conformational rearrangements preceding the ion movements. The high activation energy obtained for both processes, 42.3 kJ mol(-1) and 60.3 kJ mol(-1) at pH 7.2, support this concept. Transient binding of Ca ions to the loop L67 and a movement of the Ca-loaded loop are discussed as a mechanism that facilitates the entrance of both Ca ions into the access channel to the ion-binding sites.
Peinelt
Christine
C
Department of Biology, University of Konstanz, Konstanz, Germany.
Apell
Hans-Jürgen
HJ
D000818:Animals; D001665:Binding Sites / Q000528:radiation effects; D002118:Calcium / Q000737:chemistry*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000528:radiation effects; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding / Q000528:radiation effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D014466:Ultraviolet Rays
D000252:Calcium-Transporting ATPases; D002118:Calcium