Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide. Evidence for two carboxyl groups that are essential for enzymatic activity.
N,N'-Dicyclohexylcarbodiimide (DCCD), a reagent that reacts with carboxyl groups under mild conditions, irreversibly inhibits (Na+,K+)-ATPase activity (measured by using 1 mM ATP) with a pseudo-first-order rate constant of 0.084 min-1 (0.25 mM DCCD and 37 degrees C). The partial activities of the enzyme, including (Na+,K+)-ATPase at 1 microM ATP, Na+-ATPase, and the formation of enzyme-acyl phosphate (E-P), decayed at about one-third the rate at which (Na+,K+)-ATPase at 1 mM ATP was lost. The formation of E-P from inorganic phosphate was unaffected by DCCD while K+-phosphatase activity decayed at the same rate as (Na+,K+)-ATPase measured at 1 mM ATP. The enzyme's substrates (i.e., sodium, potassium, magnesium, and ATP) all decreased the rate of DCCD inactivation of (Na+,K+)-ATPase activity measured at either 1 mM or 1 microM ATP. The concentration dependence of the protection afforded by each substrate is consistent with its binding at a catalytically relevant site. DCCD also causes cross-linking of the enzyme into species of very high molecular weight. This process occurs at about one-tenth the rate at which (Na+,K+)-ATPase activity measured at 1 mM ATP is lost, too slowly to be related to the loss of enzymatic activity. Labeling of the enzyme with [14C]DCCD shows the incorporation of approximately 1 mol of DCCD per mole of large subunit; however, the incorporation is independent of the loss of enzymatic activity. The results presented here suggest that (Na+,K+)-ATPase contains two carboxyl groups that are essential for catalytic activity, in addition to the previously known aspartate residue which is involved in formation of E-P.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004285:Dogs; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*
D002234:Carbodiimides; D046911:Macromolecular Substances; D004024:Dicyclohexylcarbodiimide; D000254:Sodium-Potassium-Exchanging ATPase