Reaction mechanism of Ca2+ ATPase of sarcoplasmic reticulum. Equilibrium and transient study of phosphorylation with Ca.ATP as substrate.
At pH 7.0 and 5 degrees C, in the absence of potassium and magnesium, the Ca-ATPase of the sarcoplasmic reticulum slowly hydrolyzes the Ca.ATP at a rate of 0.05 s-1. During turnover 4 nmol of phosphoenzyme per mg of total protein accumulate with a Km value of 10(-8) M. Combining rapid filtration and rapid acid quenching, we took advantage of the above properties to study the early steps of phosphorylation. (formula; see text) Direct measurements by rapid filtration of the rate constant of Ca.ATP binding (k1 = 3.5 x 10(6) M-1 s-1) and dissociation (k-1 = 2.5 s-1) enable us to estimate Ca.ATP affinity (Kd = 7 x 10(-7) M). Rapid acid quenching shows that covalent phosphoenzyme forms slowly with a rate constant of 0.6 s-1 (k2), and parallel filtration experiments indicate that phosphorylation and ADP release occur simultaneously. If the reaction is reversed (toward ATP synthesis) 70% of the phosphoenzyme is ADP-sensitive and ADP-induced dephosphorylation is fast (k-2 = 15 s-1), whereas the ADP-insensitive phosphoenzyme slowly hydrolyzes in the forward direction of the ATPase cycle at a rate of 0.05 s-1. Combination of rapid filtration and rapid acid quenching and the use of a mixture of [14C]ATP and [32P]ATP allow us to study the kinetics of several transient species. These data are used to develop a molecular mechanism for Ca-ATPase phosphorylation.
Lacapere
J J
JJ
Service de Biophysique (URA 1290 Centre National de la Recherche Scientifique), Departement de Biologie, CEN Saclay, Gif sur Yvette Cedex, France.
Guillain
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013997:Time Factors
D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases