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.
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.
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