Substrate recognition at the cytoplasmic and extracellular binding site ofthe lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus catalyze s the uptake of lactose in an exchange reaction with intracellularly formed galactose, The interactions between the substrate and the cyto plasmic and extracellular binding site of LacS have been characterized by assaying bin ding and transport of a range of sugars in proteoliposomes, in which the pu rified protein was reconstituted with a unidirectional orientation. Specifi city for galactoside binding is given by the spatial configuration of the C -2, C-3, C-4, and C-6 hydroxyl groups of the galactose moiety, Except for a C-4 methoxy substitution, replacement of the hydroxyl groups for bulkier g roups is not tolerated at these positions. Large hydrophobic or hydrophilic substitutions on the galactose C-1 alpha or beta position did not impair t ransport. In fact, the hydrophobic groups increased the binding affinity bu t decreased transport rates compared with galactose, Binding and transport characteristics of deoxygalactosides from either side of the membrane showe d that the cytoplasmic and extracellular binding site interact differently with galactose, Compared with galactose, the IC50 values for 2-deoxy- and 6 -deoxygalactose at the cytoplasmic binding site were increased 150- and 20- fold, respectively, whereas they were the same at the extracellular binding site. From these and other experiments, we conclude that the binding sites and translocation pathway of LacS are spacious along the C-1 to C-4 axis o f the galactose moiety and are restricted along the C-2 to C-6 axis. The di fferences in affinity at the cytoplasmic and extracellular binding site ens ure that the transport via LacS is highly asymmetrical for the two opposing directions of translocation.