A Nontransportable Substrate for Lactose Permease

A substrate for lactose permease of Escherichia coli was synthesized that binds to the protein with a relatively high affinity, but is not transported to any detectable extent. This substrate, 6'-[(N-phenylalanylphenylalanyl)amino]hexyl 1-thio-beta-D-galactoside, is a peptide galactoside composed of a bulky aromatic dipeptide that is linked to galactose via an aminohexyl spacer. Binding of the peptide galactoside to lactose permease in cytoplasmic membranes was determined in a competition assay yielding a dissociation constant of 150 microM. Transport was measured by a counterflow assay using lipid vesicles with reconstituted lactose permease. An upper limit for the rate constant of transport was obtained as 0.02 s-1, 3 orders of magnitude smaller than the value for lactose.