A CONSERVED ASPARTATE RESIDUE, ASP187, IS IMPORTANT FOR NA-DEPENDENT PROLINE BINDING AND TRANSPORT BY THE NA+()PROLINE TRANSPORTER OF ESCHERICHIA-COLI/

Asp 187 in the Na+/proline transporter of Escherichia coli (PutP) is c onserved within the Na+/ solute cotransporter family. Information on t he role of this residue has been gained by amino acid substitution ana lysis. PutP with Glu, Asn, or Cys in place of Asp187 catalyzed Na+-cou pled proline uptake at 75%, 25%, and 1.5%, respectively, of the V-max of PutP-wild-type while the apparent K-m for proline was only slightly altered. Importantly, acetylation or amidoacetylation of an engineere d transporter containing a single Cys at position 187 stimulated proli ne uptake. Strikingly, PutP-D187C exhibited high-affinity proline bind ing even at very low Na+ concentrations (2 mu M) while proline binding to PutP-wild-type, -D187E, and -D187N was strictly dependent on the N ai concentration. The apparent independence of proline binding from th e Na+ concentration can at least partially be attributed to an enhance d Na+ affinity of PutP-D187C. In addition, reaction of PutP containing a single Cys at position 187 with N-ethylmaleimide was inhibited by N a+ 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 fo r active proline transport. It is suggested that Asp187 is located clo se 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 .