TOPOLOGY OF ALLOSTERIC REGULATION OF LACTOSE PERMEASE

Sugar transport by some permeases in Escherichia coli is allostericall y regulated by the phosphorylation state of the intracellular regulato ry protein, enzyme IIA(glc) of the phosphoenolpyruvate:sugar phosphotr ansferase system. A sensitive radiochemical assay for the interaction of enzyme IIA(glc) with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by tra nsportable substrates such as lactose, melibiose, and raffinose, but n ot by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand bindin g and transport by alkylating Cys-148, also blocks enzyme IIA(glc) bin ding. Preincubation with the substrate analog beta-D-galactopyranosyl 1-thio-beta-D-galactapyranoside protects both lactose transport and en zyme IIA(glc) binding against inhibition by ethylmaleimide. A collecti on of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIA(glc) binding. The nature of the interaction of enzyme IIA(glc) with the cytoplasmic face of lactose permease was explored. T he N- and C-termini, as well as five hydrophilic loops in the permease , are exposed on the cytoplasmic surface of the membrane and it has be en proposed that the central cytoplasmic loop of lactose permease is t he major determinant for interaction with enzyme IIA(glc). Lactose per mease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIA(glc), indicat ing that these regions of lactose permease may be involved in recognit ion of enzyme IIA(glc). Taken together, these results suggest that int eraction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optim al for interaction with the regulatory protein, enzyme IIA(glc). A top ological map of the proposed interaction is presented.