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.
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.
Tzubery
Tzvi
T
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Rimon
Abraham
A
Padan
Etana
E
D017920:Antiporters; D001426:Bacterial Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D012964:Sodium / Q000737:chemistry*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D017920:Antiporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; C102839:NhaA protein, E coli; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D014357:Trypsin; D003545:Cysteine