ArticleProceedings of the National Academy of Sciences of the United States of America2012
Role of protons in sugar binding to LacY.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 20 papers.
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Who cites it
20 citing papers in PubMed, 33 citations in OpenAlex.
- Proton reactions: From basic science to biomedical applications.Biophysical journal · 2024Article
- Structures and General Transport Mechanisms by the Major Facilitator Superfamily (MFS).Chemical reviews · 2021Article
- Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins - SWEET talking the COPs.Journal of cell science · 2020Review
- Structural and Functional Adaptability of Sucrose and Lactose Permeases fromBiochemistry · 2020Article
- The proton electrochemical gradient induces a kinetic asymmetry in the symport cycle of LacY.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- Engineered occluded apo-intermediate of LacY.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- pKProceedings of the National Academy of Sciences of the United States of America · 2017Article
- Energy coupling mechanisms of MFS transporters.Protein science : a publication of the Protein Society · 2015Review
- Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site.Proceedings of the National Academy of Sciences of the United States of America · 2015Article
- Kinetic and vibrational isotope effects of proton transfer reactions in channelrhodopsin-2.Biophysical journal · 2015Article
- Molecular insights into proton coupled peptide transport in the PTR family of oligopeptide transporters.Biochimica et biophysica acta · 2015Review
- A chemiosmotic mechanism of symport.Proceedings of the National Academy of Sciences of the United States of America · 2015Article
- Functionally important carboxyls in a bacterial homologue of the vesicular monoamine transporter (VMAT).The Journal of biological chemistry · 2014Article
- Functional architecture of MFS D-glucose transporters.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
- Structure of sugar-bound LacY.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
- Protonation drives the conformational switch in the multidrug transporter LmrP.Nature chemical biology · 2014Article
- Structure-based mechanism for Na(+)/melibiose symport by MelB.Nature communications · 2014Article
- Evolutionary mix-and-match with MFS transporters II.Proceedings of the National Academy of Sciences of the United States of America · 2013Article
- Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A.Proceedings of the National Academy of Sciences of the United States of America · 2013Article
- Trp replacements for tightly interacting Gly-Gly pairs in LacY stabilize an outward-facing conformation.Proceedings of the National Academy of Sciences of the United States of America · 2013Article
Corrections and comments
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Authors and funding
5 authors at 1 institution in 2 countries.
Funding
Abstract
WT lactose permease of Escherichia coli (LacY) reconstituted into proteoliposomes loaded with a pH-sensitive fluorophore exhibits robust uphill H(+) translocation coupled with downhill lactose transport. However, galactoside binding by mutants defective in lactose-induced H(+) translocation is not accompanied by release of an H(+) on the interior of the proteoliposomes. Because the pK(a) value for galactoside binding is ∼10.5, protonation of LacY likely precedes sugar binding at physiological pH. Consistently, purified WT LacY, as well as the mutants, binds substrate at pH 7.5-8.5 in detergent, but no change in ambient pH is observed, demonstrating directly that LacY already is protonated when sugar binds. However, a kinetic isotope effect (KIE) on the rate of binding is observed, indicating that deuterium substitution for protium affects an H(+) transfer reaction within LacY that is associated with sugar binding. At neutral pH or pD, both the rate of sugar dissociation (k(off)) and the forward rate (k(on)) are slower in D(2)O than in H(2)O (KIE is ∼2), and, as a result, no change in affinity (K(d)) is observed. Alkaline conditions enhance the effect of D(2)O on k(off), the KIE increases to 3.6-4.0, and affinity for sugar increases compared with H(2)O. In contrast, LacY mutants that exhibit pH-independent high-affinity binding up to pH 11.0 (e.g., Glu325 → Gln) exhibit the same KIE (1.5-1.8) at neutral or alkaline pH (pD). Proton inventory studies exhibit a linear relationship between k(off) and D(2)O concentration at neutral and alkaline pH, indicating that internal transfer of a single H(+) is involved in the KIE.
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