Binding of Enzyme IIAGlc, a Component of the Phosphoenolpyruvate:Sugar Phosphotransferase System, to the Escherichia coli Lactose Permease
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- 作者：Melissa Sondej ; Adam B. Weinglass ; Alan Peterkofsky ; and H. Ronald Kaback
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：April 30, 2002
- 年：2002
- 卷：41
- 期：17
- 页码：5556 - 5565
- 全文大小：156K
- 年卷期：v.41,no.17(April 30, 2002)
- ISSN：1520-4995
文摘
Enzyme IIAGlc, 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 IIAGlc 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]IIAGlc binding promoted by melibiose exhibitsan affinity (KDIIA) of 
1 
M and a stoichiometry of one mole of IIAGlc per six moles of lactose permease.Both the quantity of [125I]IIAGlc bound and the sugar concentration required for half-maximal IIAGlc binding(K0.5IIAsug) was measured for eight permease substrates. Differences in maximal IIAGlc 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. IIAGlc 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 IIAGlc 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 IIAGlc.
1 M and a stoichiometry of one mole of IIAGlc per six moles of lactose permease.Both the quantity of [125I]IIAGlc bound and the sugar concentration required for half-maximal IIAGlc binding(K0.5IIAsug) was measured for eight permease substrates. Differences in maximal IIAGlc 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. IIAGlc 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 IIAGlc 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 IIAGlc.