Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum. The role of Mg2+ that activates hydrolysis of the phosphoenzyme.
The role of Mg2+ in the activation of phosphoenzyme hydrolysis has been investigated with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. The enzyme of the native and solubilized vesicles was phosphorylated with ATP at 0 degrees C, pH 7.0, in the presence of Ca2+ and Mg2+. When Ca2+ and Mg2+ in the medium were chelated, phosphoenzyme hydrolysis continued for about 15 s and then ceased. The extent of this hydrolysis increased with increasing concentrations of Mg2+ added before the start of phosphorylation. This shows that the hydrolysis was activated by the Mg2+ added. The Mg2+ which activated phosphoenzyme hydrolysis was distinct from Mg2+ derived from MgATP bound to the substrate site. The Mg2+ site at which Mg2+ combined to activate phosphoenzyme hydrolysis was located on the outer surface of the vesicular membranes. During the catalytic cycle, Mg2+ combined with the Mg2+ site before Ca2+ dissociated from the Ca2+ transport site of the ADP-sensitive phosphoenzyme with bound Ca2+. This Mg2+ did not activate hydrolysis of the ADP-sensitive phosphoenzyme with bound Ca2+, but markedly activated hydrolysis of the ADP-insensitive phosphoenzyme without bound Ca2+. It is concluded that during the catalytic cycle, Mg2+ activates phosphoenzyme hydrolysis only after Ca2+ has dissociated from the Ca2+ transport site of phosphoenzyme.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D051379:Mice; D009132:Muscles / Q000201:enzymology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D004533:Egtazic Acid; D004492:Edetic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium