Thapsigargin and Dimethyl Sulfoxide Activate Medium Pi ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca2+-ATPase

Abstract

Thapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca2+-ATPase. It binds the Ca2+-free E2 conformation in the picomolar range, supposedly resulting in a largely catalytically inactive species. We now find that thapsigargin has little effect on medium Pi ↔ HOH oxygen exchange and that this activity is greatly stimulated (up to 30-fold) in the presence of 30% (v/v) Me2SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me2SO on the fourrate constants governing the reaction of Pi with Ca2+-ATPase. The principal effect of thapsigargin alone is to stimulate EP hydrolysis (k_2), whereas that of Me2SO is to greatly retard Pi dissociation (k_1), accounting for its well known effect on increasing the apparent affinity for Pi. These effects persist when the agents are used in combination and substantially account for the activated oxygen exchange (Vexchange = k_2[EP]). Kinetic simulations show that the overall rate constant for the formation of EP is very fast (-300 s-1) when the exchange is maximal. Thapsigargin greatly stabilizes Ca2+-ATPase against denaturation in detergent in the absence of Ca2+, as revealed by glutaraldehyde cross-linking, suggesting that the membrane helices lock together. It seems that the reactions at the phosphorylation site, associated with the activated exchange reaction, are occurring without much movement of the transport site helices, and we suggest that they may be associated solely with an occluded H+ state.