title: Sugar recognition by the lactose permease of Escherichia coli.
abstract: Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity. The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site. We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269. Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates. Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N. It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit.
authors.affiliation: Department of Physiology and Microbiology, Molecular Biology Institute, Howard Hughes Medical Institute, Los Angeles, CA 90095-1662, USA.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Vázquez-Ibar
authors.forename: Lan
authors.initials: L
authors.lastname: Guan
authors.forename: Adam B
authors.initials: AB
authors.lastname: Weinglass
authors.forename: Gill
authors.initials: G
authors.lastname: Verner
authors.forename: Ruth
authors.initials: R
authors.lastname: Gordillo
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D001665:Binding Sites; D001692:Biological Transport; D001974:Bromosuccinimide / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
chemical_list: D026901:Membrane Transport Proteins; D018698:Glutamic Acid; D014364:Tryptophan; C021499:lactose permease; D001974:Bromosuccinimide; D010100:Oxygen