Delineating electrogenic reactions during lactose/H+ symport.
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.
Garcia-Celma
Juan J
JJ
Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt/M, Germany.
Ploch
Julian
J
Smirnova
Irina
I
Kaback
H Ronald
HR
Fendler
Klaus
K
D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D055724:Electrophysiological Phenomena; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011522:Protons; D027981:Symporters / Q000737:chemistry* / Q000235:genetics
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D007785:Lactose