title: Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.
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.
authors: Department of Microbial Biotechnology, Laboratory of Applied Microbiology, Sendai 981-8555, Japan.
authors: Ayako
authors: A
authors: Sasahara
authors: Department of Biomolecular Engineering, Laboratory of Applied Biophysical Chemistry, Graduate School of Engineering, Tohoku University, Sendai, Miyagi 980-8579, and.
authors: Kei
authors: K
authors: Nanatani
authors: Department of Applied Bioorganic Chemistry, Laboratory of Applied Bioorganic Chemistry, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
authors: Masaru
authors: M
authors: Enomoto
authors: Department of Applied Bioorganic Chemistry, Laboratory of Applied Bioorganic Chemistry, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
authors: Shigefumi
authors: S
authors: Kuwahara
authors: Department of Microbial Biotechnology, Laboratory of Applied Microbiology, Sendai 981-8555, Japan; Microbial Genomics Laboratory, New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi 980-8579, Japan. Electronic address: kabe@biochem.tohoku.ac.jp.
authors: Keietsu
authors: K
authors: Abe
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology; D056569:Enterococcaceae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D008081:Liposomes; D001224:Aspartic Acid; D000409:Alanine