THE REACTION-MECHANISM OF CA2+-ATPASE OF SARCOPLASMIC-RETICULUM - DIRECT MEASUREMENT OF THE MG.ATP DISSOCIATION-CONSTANT GIVES SIMILAR VALUES IN THE PRESENCE OR ABSENCE OF CALCIUM

Combining rapid filtration and rapid acid quenching, we have directly measured, at pH 7.0 and 5-degrees-C, the association and dissociation rate constants of Mg . ATP binding to the sarcoplasmic reticulum (SR) ATPase in the presence of 50 muM calcium and 5 mM MgCl2 (3 - 4 x 10(6) M-1 . S-1 and 9 s-1, respectively). Therefore, we have determined the true affinity for Mg . ATP (K(d) = 3 muM) in the presence of calcium, which can not be measured at equilibrium because of spontaneous and fast phosphorylation. At low concentrations, Mg - ATP binding is the rate limiting step in the phosphorylation process, and Mg . ATP dissociation is slower than dephosphorylation. The kinetics of Ca2+ binding measured by rapid filtration are biphasic, reflecting a two-step mechanism, both steps being accelerated by Mg . ATP. Combining rapid filtration and rapid monitoring of the intrinsic fluorescence of SR Ca2+-ATPase, we showed that rate constants for calcium binding are always lower than those of Mg . ATP binding to an EGTA-incubated enzyme. We measured dissociation and association rate constants of Mg . ATP binding in the absence of calcium (k-1 = 25 s-1 and k1 = 7.5 10(6) M-1 - s-1). This gives a K(d) similar to that obtained by equilibrium measurements (3 - 4 muM). Both non-phosphorylated conformations of the enzyme have similar affinity for Mg . ATP. Therefore, activation of ATPase activity by an excess of ATP cannot be explained by a change in affinity of the non-phosphorylated enzyme for Mg . ATP. In conjunction with previous results, these data are used to discuss the molecular mechanism for the Ca2+-ATPase cycle, in which ATP is sequentially substrate and activator on a multiple-function single site.