PMID:14604993
| Citation |
Tzubery, T, Rimon, A and Padan, E (2004) Mutation E252C increases drastically the Km value for Na+ and causes an alkaline shift of the pH dependence of NhaA Na+/H+ antiporter of Escherichia coli. J. Biol. Chem. 279:3265-72 |
|---|---|
| Abstract |
A single Cys replacement of Glu at position 252 (E252C) in loop VIII-IX of NhaA increases drastically the Km 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 Lys(249) 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 Lys(249), 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. |
| Links |
PubMed Online version:10.1074/jbc.M309021200 |
| Keywords |
Antiporters; Bacterial Proteins/chemistry; Cell Membrane/metabolism; Cysteine/chemistry; Dose-Response Relationship, Drug; Escherichia coli/metabolism; Escherichia coli Proteins; Hydrogen-Ion Concentration; Kinetics; Ligands; Mutagenesis, Site-Directed; Mutation; Plasmids/metabolism; Protein Conformation; Protein Structure, Secondary; Protons; Sodium/chemistry; Sodium-Hydrogen Antiporter/chemistry; Sodium-Hydrogen Antiporter/genetics; Sodium-Hydrogen Antiporter/physiology; Trypsin/chemistry; Trypsin/pharmacology |
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