Interaction between the cytoplasmic and membrane-bound domains of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.
Sulfhydryl reagents affected the binding properties of the translocator domain, NIII, of enzyme IImtl in two ways: (i) the affinity for mannitol was reduced, and (ii) the exchange rate of bound and free mannitol was increased. The effect on the affinity was very much reduced after solubilization of enzyme IImtl in the detergent decylPEG. The effects were caused exclusively by reaction of the sulfhydryl reagents with the cysteine residue at position 384 in the primary sequence. Interaction between two domains is involved, since Cys384 is located in the cytoplasmic domain, CII. When Cys384 was mutated to serine, the enzyme exhibited the same binding properties as the chemically modified enzyme. The data support our proposal that phosphorylation of enzyme IImtl drastically reduces the activation energy for the translocation step through interaction between domains CII and NIII [Lolkema J. S., ten Hoeve-Duurkens, R. H., Swaving Dijkstra, D., & Robillard, G. T. (1991) Biochemistry (preceding paper in this issue)]. Functional interaction between the translocator domain, NIII, and domain CI was investigated by phosphorylation of His554, located in domain CI, in the C384S mutant. No effect on the binding properties was observed. In addition, the binding properties were insensitive to the presence of the soluble phosphotransferase components enzyme I and HPr.
Lolkema
J S
JS
Department of Physical Chemistry, University of Groningen, The Netherlands.
Dijkstra
D S
DS
ten Hoeve-Duurkens
R H
RH
Robillard
G T
GT
D003593:Cytoplasm / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli