Molecular Pharmacology, Vol 9, 51-60, Copyright © 1973 by the American Society for Pharmacology and Experimental Therapeutics

Structure-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

¹ Department of Pharmacology, University of Wisconsin Medical School, Madison , Wisconsin 53706

The dissociation rate constants (k_d) 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, k_d was dependent on the nature of the sugar and the temperature but not on the steroid. The 3'-hydroxyl and the 5'--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 3'-hydroxyl group decreased this stability. These data indicate a sugar site on the enzyme, and they suggest that the 3'-hydroxyl is bound to two groups at this site, a proton-donating group and a proton-accepting group. The 3'--hydroxyl binds to either group, but the 3'--hydroxyl and the 3'--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.

Note:
ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. Lowell E. Hokin for his interest and thoughtful help with the manuscript. He also wishes to thank Dr. Robert A. Ellison, School of Pharmacy, University of Wisconsin, for helpful discussion concerning the configuration of cardiac glycosides.