The reaction mechanism of Ca(2+)-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 microM 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 (Kd = 3 microM) 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 Ca(2+)-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 Kd similar to that obtained by equilibrium measurements (3-4 microM). 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 Ca(2+)-ATPase cycle, in which ATP is sequentially substrate and activator on a multiple-function single site.
Lacapere
J J
JJ
Département de Biologie Cellulaire et Moléculaire et URA CNRS 1290, Centre d'Etudes Nucléaires, Gif sur Yvette, France.
Guillain
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000001:Calcimycin / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002474:Cell-Free System; D004533:Egtazic Acid / Q000494:pharmacology; D005374:Filtration; D066298:In Vitro Techniques; D007700:Kinetics; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D000001:Calcimycin; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D011188:Potassium