The reaction of sulfhydryl groups of sodium and potassium ion-activated adenosine triphosphatase with N-ethylmaleimide. The relationship between ligand-dependent alterations of nucleophilicity and enzymatic conformational states.
The reaction between N-ethylmaleimide and (Na+ + K+)-ATPase, performed under ligand conditions which produce each of the kinetic states of the enzyme and their associated conformational forms, was examined through an analysis of the inhibition of enzymatic activity and the incorporation of radiolabeled reagent into the enzyme. The inactivation reactions displayed pseudo-first order kinetics with respect to the concentration of active enzyme, indicating that the loss of activity is associated with the alkylation of a unique sulfhydryl group. In the absence of enzyme phosphorylation, the nucleophilicity of this sulfhydryl group is affected primarily by the nature of the monovalent cation present and does not correlate with the conformational state. A method for determining the actual concentration and specific radioactivity of radiolabeled N-ethylmaleimide during the reaction with (Na+ + K+)-ATPase was developed, allowing the measurement of the total reactive sulfhydryl groups of native (Na+ + K+)-ATPase under conditions identical with those of the inactivation studies. The labeling of the enzyme complex is associated almost exclusively with the large polypeptide, which contains four sulfhydryl groups which react with this reagent. One of these residues is presumably the sulfhydryl responsible for inactivation of the enzyme. Two react stoichiometrically and rapidly with N-ethylmaleimide under all conditions. The nucleophilicity of the fourth sulfhydryl group is governed by the conformational state of the enzyme, but the alkylation of this residue does not result in loss of enzymatic activity.
D000818:Animals; D001665:Binding Sites; D004285:Dogs; D005033:Ethylmaleimide / Q000494:pharmacology*; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013438:Sulfhydryl Compounds
D008024:Ligands; D013438:Sulfhydryl Compounds; D000254:Sodium-Potassium-Exchanging ATPase; D005033:Ethylmaleimide