Binding of Hydrophobic d-Galactopyranosides to the Lactose Permease of Escherichia coli^†

Binding of α- and β-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 α-d-galactopyranosides exhibit a 60-fold increase in binding affinity (K_D = 0.5 mM), relative to galactose (K_D = 30 mM), while methyl β-d-galactopyranoside binds only 3-fold better. In the present study, galactopyranosides with cyclohexyl or phenyl substitutions, both in α and β anomeric configurations, were synthesized. Surprisingly, relative to methyl α-d-galactopyranoside, binding of cyclohexyl α-d-galactopyranoside to lactose permease is essentially unchanged (K_D = 0.4 mM), and phenyl α-d-galactopyranoside exhibits only a modest increase in binding affinity (K_D = 0.15 mM). Nitro- or methyl-substituted phenyl α-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 β 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 α-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 α- or β-d-galactopyranosides thus may provide a rationale for the characteristic binding preference of the permease for α anomers.