Structue-activity relationships of cardiotonic steroids for the inhibition of sodium- and potassium-dependent adenosine triphosphatase. I. Dissociation rate constants of various enzyme-cardiac glycoside complexes formed in the presence of magnesium and phosphate.

Abstract

The dissociation rate constants (kd) of cardiac monoglycoside-(Na+ + K+)-ATPase complexes in the presence of magnesium and inorganic phosphate were determined by enzymatic assay after dilution. Among various cardiac monoglycosides, kd was dependent on the nature of the sugar and the temperature but not on the steroid. The 39-hydroxyl and the 59-α-methyl group of the sugar markedly influenced the stability of the complex. The order of stability of cardiac monoglycoside-(Na+ + K+)-ATPase complexes is the following: L-rhamnoside > D-6-deoxyguloside [unknown] D-digitoxide > D-fucoside [unknown] D-6-deoxyglucoside. Methylation or acetylation of the 39-hydroxyl group decreased this stability. These data indicate a sugar site on the enzyme, and they suggest that the 39-hydroxyl is bound to two groups at this site, a proton-donating group and a proton-accepting group. The 39-α-hydroxyl binds to either group, but the 39-β-hydroxyl and the 39-α-methoxyl bind only a proton-accepting group and a proton-donating group, respectively. The activation energy of this dissociation was rather constant (20 kcal/mole) with various cardiac monoglycosides. The rate-determining step of the dissociation might be a conformational change of the enzyme, and it is suggested that the reaction order is the following: dissociation of the sugar portion of the cardiac glycoside from the sugar site of enzyme, conformational change of the sugar site, and dissociation of the steroid portion from the steroid site.