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A nontransportable substrate for lactose permease

C Seibert1, W Dörner, F Jähnig

  • 1Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Tübingen, Germany.

Biochemistry
|June 20, 1995
PubMed
Summary

Researchers synthesized a novel peptide galactoside that binds to Escherichia coli lactose permease but is not transported. This non-transported substrate offers insights into lactose permease function and substrate recognition mechanisms.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Microbiology

Background:

  • Lactose permease (LacY) is a key transporter in Escherichia coli, crucial for lactose metabolism.
  • Understanding LacY's transport mechanism requires specific substrate analogs to probe binding and translocation.
  • Existing substrates do not fully elucidate the intricate steps of LacY-mediated transport.

Purpose of the Study:

  • To synthesize and characterize a novel non-transported substrate for lactose permease.
  • To investigate the binding affinity and transport kinetics of this synthetic peptide galactoside.
  • To gain deeper insights into the structure-function relationship of lactose permease.

Main Methods:

  • Chemical synthesis of 6'-[(N-phenylalanylphenylalanyl)amino]hexyl 1-thio-beta-D-galactoside.
  • Competition binding assays using purified LacY in cytoplasmic membranes.
  • Counterflow transport assays with reconstituted proteoliposomes containing purified LacY.

Main Results:

  • The synthesized peptide galactoside exhibited high-affinity binding to lactose permease (Kd = 150 microM).
  • Transport of the substrate was undetectable, with an upper limit for the rate constant of 0.02 s-1.
  • The transport rate was significantly lower (3 orders of magnitude) compared to natural lactose.

Conclusions:

  • The synthetic peptide galactoside acts as a high-affinity binder but a non-transporter for lactose permease.
  • This finding highlights the distinct structural requirements for binding versus translocation in LacY.
  • The study provides a valuable tool for dissecting the mechanism of sugar transport by LacY.

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