title: Binding of hydrophobic D-galactopyranosides to the lactose permease of Escherichia coli.
abstract: Binding of alpha- and beta-D-galactopyranosides with different hydrophobic aglycons was compared using substrate protection against N-ethylmaleimide alkylation of single-Cys148 lactose permease. As demonstrated previously, methyl- or allyl-substituted alpha-D-galactopyranosides exhibit a 60-fold increase in binding affinity (K(D) = 0.5 mM), relative to galactose (K(D) = 30 mM), while methyl beta-D-galactopyranoside binds only 3-fold better. In the present study, galactopyranosides with cyclohexyl or phenyl substitutions, both in alpha and beta anomeric configurations, were synthesized. Surprisingly, relative to methyl alpha-D-galactopyranoside, binding of cyclohexyl alpha-D-galactopyranoside to lactose permease is essentially unchanged (K(D) = 0.4 mM), and phenyl alpha-D-galactopyranoside exhibits only a modest increase in binding affinity (K(D) = 0.15 mM). Nitro- or methyl-substituted phenyl alpha-D-galactopyranosides bind with significantly higher affinities (K(D) = 0.014-0.067 mM), and the strongest binding is observed with analogues containing para substituents. In contrast, D-galactopyranosides with a variety of large hydrophobic substituents (isopropyl, cyclohexyl, phenyl, o- or p-nitrophenyl) in beta anomeric configuration exhibit uniformly weak binding (K(D) = 1.0-2.3 mM). The results confirm and extend previous observations that hydrophobic aglycons of D-galactopyranosides increase binding affinity, with a clear predilection toward alpha-substituted sugars. In addition, the data suggest that the primary interaction between the permease and hydrophobic aglycons is directed toward the carbon atom bonded to the anomeric oxygen. The different positioning of this carbon atom in alpha- or beta-D-galactopyranosides thus may provide a rationale for the characteristic binding preference of the permease for alpha anomers.
authors.affiliation: Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles, California 90095-1662, USA.
authors.forename: Miklós
authors.initials: M
authors.lastname: Sahin-Tóth
authors.forename: Paula
authors.initials: P
authors.lastname: Gunawan
authors.forename: Mary C
authors.initials: MC
authors.lastname: Lawrence
authors.forename: Tatsushi
authors.initials: T
authors.lastname: Toyokuni
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D001665:Binding Sites; D002135:Calcium-Binding Proteins; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005690:Galactose / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008756:Methylgalactosides / Q000737:chemistry / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism; D009598:Nitrophenylgalactosides / Q000138:chemical synthesis / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism; D027981:Symporters; D013862:Thiogalactosides / Q000737:chemistry / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D027981:Symporters; D013862:Thiogalactosides; C023550:galactose-binding protein; C009468:thiomethylgalactoside; C031552:methyl beta-galactoside; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; C021499:lactose permease; D005690:Galactose