A conserved aspartate residue, Asp187, is important for Na+-dependent proline binding and transport by the Na+/proline transporter of Escherichia coli.
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.
Quick
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M
Universität Osnabrück, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie, Germany.
Jung
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H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D026921:Amino Acid Transport Systems, Neutral; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D001693:Biological Transport, Active; D015153:Blotting, Western; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism; D007460:Iodoacetamide; D019807:Iodoacetic Acid; D007700:Kinetics; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011392:Proline / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*
D026921:Amino Acid Transport Systems, Neutral; D008024:Ligands; D026901:Membrane Transport Proteins; D001224:Aspartic Acid; C033082:proline transporter; D011392:Proline; D012964:Sodium; D003545:Cysteine; D019807:Iodoacetic Acid; D007460:Iodoacetamide