Cooperative Binding of the Sugar Substrates and Allosteric Regulatory Protein (Enzyme III Glc of the Phosphotransferase System) to the Lactose and Melibiose Permeases in Escherichia coli and Salmonella typhimurium

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Abstract
An Escherichia coli strain which overproduces the lactose permease was used to investigate the mechanism of allosteric regulation of this permease and those specific for melibiose, glycerol, and maltose by the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Thio-β-digalactoside, a high affinity substrate of the lactose permease, released the glycerol and maltose permeases from inhibition by methyl-α-d-glucoside. Resumption of glycerol uptake occurred immediately upon addition of the galactoside. The effect was not observed in a strain which lacked or contained normal levels of the lactose permease, but growth of wild-type E. coli in the presence of isopropyl-β-thiogalactoside plus cyclic AMP resulted in enhanced synthesis of the lactose permease so that galactosides relieved inhibition of glycerol uptake. Thiodigalactoside also relieved the inhibition of glycerol uptake caused by the presence of other PTS substrates such as fructose, mannitol, glucose, 2-deoxyglucose, and 5-thioglucose. Inhibition of adenylate cyclase activity by methyl-α-glucoside was also relieved by thiodigalactoside in E. coli T52RT provided that the lactose permease protein was induced to high levels. Cooperative binding of sugar and enzyme IIIGlc to the melibiose permease in Salmonella typhimurium was demonstrated, but no cooperativity was noted with the glycerol and maltose permeases. These results are consistent with a mechanism of PTS-mediated regulation of the lactose and melibiose permeases involving a fixed number of allosteric regulatory proteins (enzyme IIIGlc) which may be titrated by the increased number of substrate-activated permease proteins. This work suggests that the cooperativity in the binding of sugar substrate and enzyme IIIGlc to the permease, demonstrated previously in in vitro experiments, has mechanistic significance in vivo. It substantiates the conclusion that PTS-mediated regulation of non-PTS permease activities involves direct allosteric interaction between the permeases and enzyme IIIGlc, the postulated regulatory protein of the PTS.