PMID:20568736
| Citation |
Garcia-Celma, JJ, Ploch, J, Smirnova, I, Kaback, HR and Fendler, K (2010) Delineating electrogenic reactions during lactose/H+ symport. Biochemistry 49:6115-21 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC2907097 Online version:10.1021/bi100492p |
| Keywords |
Binding Sites; Cell Membrane/chemistry; Cell Membrane/enzymology; Electrophysiological Processes; 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 |
| edit table |
Significance
First assignment of electrogenic steps (partial reactions that displace electric charge) in the LacY reaction cycle.
Useful Materials and Methods
This study uses an unconventional electrophysiological technique based on solid supported membranes. For details see [1]
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