Kinetics of the Ca(2+), H(+), and Mg(2+) interaction with the ion-binding sites of the SR Ca-ATPase.
Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca(2+) and H(+) on binding of the other ion in the E(1) and P-E(2) states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg(2+) a strictly competitive binding sequence, H(2)E(1) <==> HE(1) <==> E(1) <==> CaE(1) <==> Ca(2)E(1), was found with two Ca(2+) ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K(1/2)(2 Ca) = 34 nM, K(1/2)(H) = 1 nM and K(1/2)(H(2)) = 1.32 microM. Up to 2 Mg(2+) ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K(1/2)(Mg) = 165 microM, K(1/2)(Mg(2)) = 7.4 mM). In the P-E(2) state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca(2+) and H(+) binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E(2)CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca(2+) (0.4 mM and 25 mM) and H(+) (2 microM and 10 microM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E(1) and P-E(2) conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position.
Peinelt
Christine
C
Department of Biology, University of Konstanz, 78457 Konstanz, Germany.
Apell
Hans-Jürgen
HJ
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008962:Models, Theoretical; D018482:Muscle, Skeletal / Q000201:enzymology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D007477:Ions; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium