Effects of free radicals on partial reactions of the Na,K-ATPase.

The function of the Na,K-ATPase is known to be considerably impaired in the presence of free radicals such as OH.. While previous experiments were largely based on the loss of enzymatic activity of the protein, this is the first communication dealing with partial reactions of the pump cycle in the presence of free radicals produced by water radiolysis. Three different system states, which are directly involved in ion transfer catalyzed by the enzyme, showed similar sensitivity to free radical action. This is indicated by largely identical D37-doses of the decay of the reaction amplitudes investigated. The decrease in the efficiency of the enzyme functions was largely due to a lethal damage of pump molecules. A kinetic analysis of the ATP-induced conformational transition E1-->E2 revealed, however, that a minor component of the inactivation is due to a reduction of the transition rate constant. The decrease of the enzymatic activity could be simulated by the decay of the rate-limiting conformational transition. This finding indicates the conservation of a close coupling between ATP-hydrolysis and sodium translocation process throughout free-radical induced inactivation. As a result of the tight coupling, enzyme modification at different system states leads to similar functional consequences for the protein.