Mutation E252C Increases Drastically the K_m Value for Na ⁺ and Causes an Alkaline Shift of the pH Dependence of NhaA Na ⁺/H⁺ Antiporter of Escherichia coli

A single Cys replacement of Glu at position 252 (E252C) in loop VIII-IX of NhaA increases drastically the K_m for Na⁺ (50-fold) of the Na⁺/H⁺ antiporter activity of NhaA and shifts the pH dependence of NhaA activity, by one pH unit, to the alkaline range. In parallel, E252C causes a similar alkaline pH shift to the pH-induced conformational change of loop VIII-IX. Thus, although both the Na⁺/H⁺ antiporter activity of wild type NhaA and its accessibility to trypsin at position Lys²⁴⁹ in loop VIII-IX increase with pH between pH 6.5 and 7.5, the response of E252C occurs above pH 8. Furthermore, probing accessibility of pure E252C protein in dodecyl maltoside solution to 2-(4′-maleimidylanilino)-naphthalene-6-sulfonic acid revealed that E252C itself undergoes a pH-dependent conformational change, similar to position Lys²⁴⁹, and the rate of the pH-induced conformational change is increased specifically by the presence of Na⁺ or Li⁺, the specific ligands of the antiporter. Chemical modification of E252C by N-ethylmaleimide, 2-(4′-maleimidylanilino)-naphthalene-6-sulfonic acid; [2-(trimethylammonium)ethyl]methane thiosulfonate, or (2-sulfonatoethyl)methanethiosulfonate reversed, to a great extent, the pH shift conferred by E252C but had no effect on the K_m of the mutant antiporter.