Interactions of dihydroxybenzenes with the Ca(2+)-ATPase: separate binding sites for dihydroxybenzenes and sesquiterpene lactones.

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ) and other hydrophobic 1,4-dihydroxybenzenes. Inhibitory potency increases on increasing substituent chain length from 2,5-dipropyl-1,4-dihydroxybenzene to 2,5-di-tert-amyl-1,4-dihydroxybenzene, the most potent inhibitor, but then decreases for 2,5-bis(7-methylheptyl)-1,4-dihydroxybenzene. Kinetic measurements are consistent with isomerization following the initial binding of BHQ to the ATPase to give a modified E2 conformation, E2AI, as for the binding of sesquiterpene lactones, such as thapsigargin. Binding of BHQ to the ATPase shifts the E1-E2 equilibrium toward E2 because of the formation of E2AI. Measurements of Ca2+ binding as a function of BHQ concentration suggest that BHQ can bind to the E1 conformation of the ATPase (but without the subsequent conformational change observed on binding to E2) and that the binding constants of E1 for Ca2+ are unaffected by binding of BHQ. Binding of BHQ to the ATPase in the presence of substoichiometric amounts of thapsivillosin A and effects of mixtures of BHQ and thapsivillosin A show that these two inhibitors have separate binding sites on the ATPase.