Enzyme IIA
Glc, encoded by the
crr gene of the phosphoenolpyruvate:sugar phosphotransferasesystem, plays an important role in regulating intermediary metabolism in
Escherichia coli ("cataboliterepression"). One function involves inhibition of inducible transport systems ("inducer exclusion"), andwith lactose permease, a galactoside is required for unphosphorylated IIA
Glc binding to cytoplasmic loopsIV/V and VI/VII [Sondej, M., Sun, J. et al. (1999)
Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530]. Withinside-out membrane vesicles containing the permease, [
125I]IIA
Glc binding promoted by melibiose exhibitsan affinity (
KDIIA) of
1
M and a stoichiometry of one mole of IIA
Glc per six moles of lactose permease.Both the quantity of [
125I]IIA
Glc bound and the sugar concentration required for half-maximal IIA
Glc binding(
K0.5IIAsug) was measured for eight permease substrates. Differences in maximal IIA
Glc binding are observed,and the
K0.5IIAsug does not correlate with the affinity of LacY for sugar. Furthermore,
K0.5IIAsug does notcorrelate with sugar affinities for various permease mutants. IIA
Glc does not bind to a mutant (Cys154
Gly), which is
locked in an outwardly facing conformation, binds with increased stoichiometry to mutantLys131
Cys, and binds only weakly to two other mutants which appear to be predominantly in eitheran outwardly or an inwardly facing conformation. When the latter two mutations are combined, sugar-dependent IIA
Glc binding returns to near wild-type levels. The findings suggest that binding of varioussubstrates to lactose permease results in a collection of unique conformations, each of which presents aspecific surface toward the inner face of the membrane that can interact to varying degrees with IIA
Glc.