Substrate specificity of the aspartate:Alanine antiporter (AspT) of tetragenococcus halophilus in reconstituted liposomes

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Abstract

The aspartate: alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate: alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate 1- with L-alanine 0. Although physiological functions of AspT were well studied, L-aspartate 1-:L-alanine 0 antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His 6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K m = 0.35 ± 0.03 mM for L-aspartate, K m ± 0.098 ± 0 mM for D-aspartate, K m±26±2 mM for L-alanine, K m=3.3 ± 0.2 mM for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.

Original languageEnglish
Pages (from-to)29044-29052
Number of pages9
JournalJournal of Biological Chemistry
Volume286
Issue number33
DOIs
Publication statusPublished - 2011 Aug 19

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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