title: Rate constants of sugar transport through two LamB mutants of Escherichia coli: comparison with wild-type maltoporin and LamB of Salmonella typhimurium.
abstract: Two LamB (maltoporin) point mutants of Escherichia coli (R8H and Y118F) and wild-type LamB of Salmonella typhimurium were reconstituted into artificial lipid bilayer membranes. Ion transport through wild-type LamB of S. typhimurium and the LamB mutants was inhibited by the addition of carbohydrates of maltose and maltooligosaccharide type in a dose-dependent fashion. The sugar-induced block of the channel function could be used for the study of current noise through the different wild-type and mutant LamB-channels. The analysis of the power density spectra allowed the evaluation of the on and off-reactions (k1 and k-1) of sugar-binding to the binding site inside the channels. Wild-type LamB of S. typhimurium had approximately the same sugar-binding kinetics as has been observed for LamB of E. coli. The results suggest that the binding site inside the channel interacts with a maximum of three glucose residues within the maltooligosaccharides. The LamB mutants R8H and Y118F showed kinetics for sugar binding substantially different from that of wild-type LamB. In particular, the on-rate, k1, for the binding of different sugars of the maltooligosaccharide series to the mutant R8H was approximately 500-times smaller than for wild-type LamB, which resulted in substantially smaller stability constant of sugar binding to the channel. Similarly, the off-rate constant, k-1, for sugar binding to the mutant Y118F decreased about 20-fold, which led to a strong increase of the affinity of carbohydrates to the site. The role of the amino residues acid R8 and Y118 in the transport of maltose and maltooligosaccharides through LamB-channels is discussed on the basis of the net flux of sugars through the channels.
authors.affiliation: Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) derUniversität Wurzburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Jordy
authors.forename: C
authors.initials: C
authors.lastname: Andersen
authors.forename: K
authors.initials: K
authors.lastname: Schülein
authors.forename: T
authors.initials: T
authors.lastname: Ferenci
authors.forename: R
authors.initials: R
authors.lastname: Benz
mesh_terms: D000473:Alkanes / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001692:Biological Transport; D004594:Electrophysiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism* / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D000473:Alkanes; D001425:Bacterial Outer Membrane Proteins; D008081:Liposomes; D009844:Oligosaccharides; D010713:Phosphatidylcholines; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; D008320:Maltose; C012867:decane