Quasi-irreversible inactivation of the sarcoplasmic reticulum Ca 2+-ATPase by simultaneous tight binding of magnesium and fluoride to the catalytic site

Abstract

The sarcoplasmic reticulum Ca 2+-ATPase was inactivated quasi-irreversibly by the treatment with KF in the presence of Mg 2+ and absence of Ca 2+. This inactivation was Mg 2+-dependent, and prevented by high-affinity Ca 2+ 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 Mg 2+ in this inactivation was in agreement with that for Mg 2+ 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 Mg 2+ binding sub-site in the catalytic site of the inactivated enzyme is occupied by tightly-bound Mg 2+. 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 Mg 2+ and F - in the inactivated enzyme were determined after unbound Mg 2+ and F - were removed by gel filtration. 2.3 mol of Mg 2+ 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 Ca 2+ binding. Linear relationships were found between the contents of the tightly-bound ligands and the extent of the enzyme inactivation. The tightly-bound Mg 2+ and F - were entirely released by low-affinity Ca 2+ binding, and correspondingly the ATPase activity was restored. It is concluded that the observed enzyme inactivation is caused by simultaneous tight binding of Mg 2+ and F - to the catalytic site.