The reaction mechanism of Ca2+‐ATPase of sarcoplasmic reticulum

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Abstract
Combining rapid filtration and rapid acid quenching, we have directly measured, at pH 7.0 and 5°C, the association and dissociation rate constants of Mg · ATP binding to the sarcoplasmic reticulum (SR) ATPase in the presence of 50 μM calcium and 5 mM MgCl2 (3–4x 106 M−1· s−1 and 9 s−1 respectively). Therefore, we have determined the true affinity for Mg · ATP (Kd= 3 μM) 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= 7.5 106 M−1· s−1). This gives a Kd similar to that obtained by equilibrium measurements (3–4 μM).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‐phophorylated 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.