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  Delineating Electrogenic Reactions during Lactose/H+ Symport

Garcia-Celma, J. J., Ploch, J., Smirnova, I., Kaback, H. R., & Fendler, K. (2010). Delineating Electrogenic Reactions during Lactose/H+ Symport. Biochemistry, 49(29), 6115-6121. doi:10.1021/bi100492p.

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 Creators:
Garcia-Celma, Juan J.1, Author           
Ploch, Julian1, Author           
Smirnova, Irina2, Author
Kaback, H. Ronald2, 3, 4, Author
Fendler, Klaus1, Author           
Affiliations:
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              
2Department of Physiology, University of California, Los Angeles, California 90095-7327, USA, ou_persistent22              
3Department of Microbiology and Immunology, University of California, Los Angeles, California 90095-7327, USA, ou_persistent22              
4Department of Molecular Genetics, University of California, Los Angeles, California 90095-7327, USA, ou_persistent22              

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Free keywords: Kinetic parameters; Sensors; Carbohydrates; Kinetic modeling; Electrical transport
 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 ~200 s-1 for wildtype 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 ~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.

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Language(s): eng - English
 Dates: 2010-06-172010-04-012010-07-022010-07-27
 Publication Status: Issued
 Pages: 7
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/bi100492p
PMID: 20568736
PMC: PMC2907097
 Degree: -

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Title: Biochemistry
Source Genre: Journal
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Affiliations:
Publ. Info: Columbus, Ohio : American Chemical Society
Pages: - Volume / Issue: 49 (29) Sequence Number: - Start / End Page: 6115 - 6121 Identifier: ISSN: 0006-2960
CoNE: https://pure.mpg.de/cone/journals/resource/954925384103