QUASI-IRREVERSIBLE INACTIVATION OF THE SARCOPLASMIC-RETICULUM CA2+-ATPASE BY SIMULTANEOUS TIGHT-BINDING OF MAGNESIUM AND FLUORIDE TO THE CATALYTIC SITE

The sarcoplasmic reticulum Ca2+-ATPase was inactivated quasi-irreversibly by the treatment with KF in the presence of Mg2+ and absence of Ca2+. This inactivation was Mg2+-dependent, and prevented by high-affinity Ca2+ binding. The enzyme was completely protected by ATP against the inactivation with an affinity consistent with that of the catalytic site for ATP. The affinity for Mg2+ in this inactivation was in agreement with that for Mg2+ in phosphorylation of the enzyme with P(i). Mg . ATP did not bind to the inactivated enzyme, whereas metal-free ATP did bind to it with a high affinity. These findings suggest that the Mg2+ binding sub-site in the catalytic site of the inactivated enzyme is occupied by tightly-bound Mg2+. The enzyme was completely protected by P(i) against the inactivation with an affinity consistent with that of the catalytic site for P(i). The inactivated enzyme showed neither phosphorylation with P(i) nor high-affinity vanadate binding. These findings suggest that the phosphorylation site of the inactivated enzyme is occupied by tightly-bound F-. The contents of tightly-bound Mg2+ and F- in the inactivated enzyme were determined after unbound Mg2+ and F- were removed by gel filtration. 2.3 mol of Mg2+ and 3.7 mol of F- per mol of phosphorylation sites were tightly bound to the enzyme. The tight binding of these ligands depended on the presence of each other, and was completely prevented by high-affinity Ca2+ binding. Linear relationships were found between the contents of the tightly-bound ligands and the extent of the enzyme inactivation. The tightly-bound Mg2+ and F- were entirely released by low-affinity Ca2+ binding, and correspondingly the ATPase activity was restored. It is concluded that the observed enzyme inactivation is caused by simultaneous tight binding of Mg2+ and F- to the catalytic site.