Delineating electrogenic reactions during lactose/H+ symport

Biochemistry. 2010 Jul 27;49(29):6115-21. doi: 10.1021/bi100492p.


Electrogenic reactions accompanying downhill lactose/H(+) symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H(+) translocation is abolished either by acidic pH or by a Glu325 --> Ala mutation in the H(+) binding site (this step with a rate constant of approximately 200 s(-1) for wild-type LacY leads to an intermediate proposed to represent an "occluded" state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of approximately 30 s(-1) and a pK of 7.5. This partial reaction is assigned to rate-limiting H(+) release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical-biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding Sites
  • Cell Membrane / chemistry
  • Cell Membrane / enzymology
  • Electrophysiological Phenomena
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Hydrogen-Ion Concentration
  • Lactose / chemistry*
  • Monosaccharide Transport Proteins / chemistry*
  • Monosaccharide Transport Proteins / genetics
  • Mutation
  • Protons
  • Symporters / chemistry*
  • Symporters / genetics


  • Escherichia coli Proteins
  • LacY protein, E coli
  • Monosaccharide Transport Proteins
  • Protons
  • Symporters
  • Lactose