pKaof Glu325 in LacY.
Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and a H+across the cytoplasmic membrane ofEscherichia coli(galactoside/H+symport). One of the most important aspects of the mechanism is the relationship between protonation and binding of the cargo galactopyranoside. In this regard, it has been shown that protonation is required for binding. Furthermore when galactoside affinity is measured as a function of pH, an apparent pK (pKapp) of ∼10.5 is obtained. Strikingly, when Glu325, a residue long known to be involved in coupling between H+and sugar translocation, is replaced with a neutral side chain, the pH effect is abolished, and high-affinity binding is observed until LacY is destabilized at alkaline pH. In this paper, infrared spectroscopy is used to identify Glu325 in situ. Moreover, it is demonstrated that this residue exhibits a pKaof 10.5 ± 0.1 that is insensitive to the presence of galactopyranoside. Thus, it is apparent that protonation of Glu325 specifically is required for effective sugar binding to LacY.
Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg CNRS, 67081 Strasbourg, France.
Natalia
N
Grytsyk
Department of Physiology, University of California, Los Angeles, CA 90095-7327.|Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, CA 90095-1570.
Junichi
J
Sugihara
Department of Physiology, University of California, Los Angeles, CA 90095-7327; rkaback@mednet.ucla.edu hellwig@unistra.fr.|Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, CA 90095-1570.|Molecular Biology Institute, University of California, Los Angeles, CA 90095-1570.
H Ronald
HR
Kaback
Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg CNRS, 67081 Strasbourg, France; rkaback@mednet.ucla.edu hellwig@unistra.fr.
Petra
P
Hellwig
D019943:Amino Acid Substitution; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009598:Nitrophenylgalactosides / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D027981:Symporters / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D054462:p21-Activated Kinases
D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D011522:Protons; D011994:Recombinant Proteins; D027981:Symporters; D009598:Nitrophenylgalactosides; D018698:Glutamic Acid; D054462:p21-Activated Kinases; D005690:Galactose
lactose permease; membrane proteins; protonation; surface-enhanced infrared spectroscopy; transport