A NONTRANSPORTABLE SUBSTRATE FOR LACTOSE PERMEASE

A substrate for lactose permease of Escherichia coli was synthesized t hat binds to the protein with a relatively high affinity, but is not t ransported to any detectable extent. This substrate, 6'-[(N-phenylalan ylphenylalanyl)amino]hexyl 1-thio-beta-D-galactoside, is a peptide gal actoside composed of a bulky aromatic dipeptide that is linked to gala ctose via an aminohexyl spacer. Binding of the peptide galactoside to lactose permease in cytoplasmic membranes was determined in a competit ion assay yielding a dissociation constant of 150 mu M. Transport was measured by a counterflow assay using lipid vesicles with reconstitute d 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 val ue for lactose.