title: The C-4 hydroxyl group of galactopyranosides is the major determinant for ligand recognition by the lactose permease of Escherichia coli.
abstract: Binding specificity in lactose permease toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by nonspecific hydrophobic interactions with the non-galactosyl moiety [Sahin-Tóth, M., Akhoon, K. M., Runner, J., and Kaback, H. R. (2000) Biochemistry 39, 5097-5103]. To characterize the contribution of individual hydroxyls, binding of structural analogues of p-nitrophenyl alpha-D-galactopyranoside (NPG) was examined by site-directed N-[(14)C]ethylmaleimide (NEM) labeling of the substrate-protectable Cys148 in the binding site. NPG blocks NEM alkylation of Cys148 with an apparent affinity of approximately 14 microM. A deoxy derivative at position C-2 binds with 25-fold lower affinity (K(D) 0.35 mM), and the deoxy analogue at C-3 exhibits ca. 70-fold decreased binding (K(D) 1 mM), while binding of 6-deoxy-NPG is at least 130-fold diminished (K(D) 1.9 mM). Remarkably, the C-4 deoxy derivative of NPG binds with almost 1500-fold reduced affinity (K(D) approximately 20 mM). No significant substrate protection is afforded by NPG analogues with methoxy (CH(3)-O-) substitutions at positions C-3, C-4, and C-6. In contrast, the C-2 methoxy analogue binds almost normally (K(D) 26 microM). The results confirm and extend the observations that the C-2, C-3, C-4, and C-6 OH groups of galactopyranosides participate in important H-bonding interactions. Moreover, the C-4 hydroxyl is identified as the major determinant of ligand binding, suggesting that sugar recognition in lactose permease may have evolved to discriminate primarily between gluco- and galactopyranosides.
authors.affiliation: Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
authors.forename: M
authors.initials: M
authors.lastname: Sahin-Tóth
authors.forename: M C
authors.initials: MC
authors.lastname: Lawrence
authors.forename: T
authors.initials: T
authors.lastname: Nishio
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005690:Galactose / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins; D011485:Protein Binding; D027981:Symporters
chemical_list: D000596:Amino Acids; D002241:Carbohydrates; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D005690:Galactose