A conserved aspartate residue, Asp187, is important for Na+-dependent proline binding and transport by the Na+/proline transporter of Escherichia coli.

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  • Author(s): Quick M;Quick M; Jung H
  • Source:
    Biochemistry [Biochemistry] 1998 Sep 29; Vol. 37 (39), pp. 13800-6.
  • Publication Type:
    Comparative Study; Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: American Chemical Society Country of Publication: United States NLM ID: 0370623 Publication Model: Print Cited Medium: Print ISSN: 0006-2960 (Print) Linking ISSN: 00062960 NLM ISO Abbreviation: Biochemistry Subsets: MEDLINE
    • Publication Information:
      Original Publication: Washington, American Chemical Society.
    • Subject Terms:
      Amino Acid Transport Systems, Neutral* ; Conserved Sequence*; Aspartic Acid/*metabolism ; Membrane Transport Proteins/*metabolism ; Proline/*metabolism ; Sodium/*metabolism; Amino Acid Sequence ; Amino Acid Substitution ; Aspartic Acid/chemistry ; Biological Transport, Active ; Blotting, Western ; Cysteine/metabolism ; Iodoacetamide ; Iodoacetic Acid ; Kinetics ; Ligands ; Membrane Transport Proteins/chemistry ; Molecular Sequence Data ; Protein Binding ; Sequence Homology, Amino Acid
    • Abstract:
      Asp187 in the Na+/proline transporter of Escherichia coli (PutP) is conserved within the Na+/solute cotransporter family. Information on the role of this residue has been gained by amino acid substitution analysis. PutP with Glu, Asn, or Cys in place of Asp187 catalyzed Na+-coupled proline uptake at 75%, 25%, and 1.5%, respectively, of the Vmax of PutP-wild-type while the apparent Km for proline was only slightly altered. Importantly, acetylation or amidoacetylation of an engineered transporter containing a single Cys at position 187 stimulated proline uptake. Strikingly, PutP-D187C exhibited high-affinity proline binding even at very low Na+ concentrations (2 microM) while proline binding to PutP-wild-type, -D187E, and -D187N was strictly dependent on the Na+ concentration. The apparent independence of proline binding from the Na+ concentration can at least partially be attributed to an enhanced Na+ affinity of PutP-D187C. In addition, reaction of PutP containing a single Cys at position 187 with N-ethylmaleimide was inhibited by Na+ but not by Li+ or proline. The results indicate that electrostatic interactions of the amino acid side chain at position 187 in PutP with other parts of the transporter and/or the coupling ion are crucial for active proline transport. It is suggested that Asp187 is located close to the pathway of the coupling ion through the membrane and may be involved in the release of Na+ on the cytoplasmic side of the membrane.
    • Accession Number:
      0 (Amino Acid Transport Systems, Neutral)
      0 (Ligands)
      0 (Membrane Transport Proteins)
      30KYC7MIAI (Aspartic Acid)
      9040-98-6 (proline transporter)
      9DLQ4CIU6V (Proline)
      9NEZ333N27 (Sodium)
      K848JZ4886 (Cysteine)
      WF5188V710 (Iodoacetic Acid)
      ZRH8M27S79 (Iodoacetamide)
    • Publication Date:
      Date Created: 19980930 Date Completed: 19981028 Latest Revision: 20131121
    • Publication Date:
      20240829
    • Accession Number:
      10.1021/bi980562j
    • Accession Number:
      9753469