Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIAGlc

Abstract

In a variety of bacteria, the phosphotransferase protein IIAGlcplays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) ofEscherichia colicatalyzes stoichiometric symport of a galactoside with an H+, using a mechanism in which sugar- and H+-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIAGlc(inducer exclusion). Here we report the thermodynamic features of the IIAGlc–LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIAGlcbinds to LacY with aKdof about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIAGlcbinding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIAGlcblocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIAGlcbinding. The precise mechanism of the inhibition of LacY by IIAGlcelucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIAGlc.