Analytical sedimentation of the IIAChb and IIBChb proteins of the Escherichia coli N,N'-diacetylchitobiose phosphotransferase system. Demonstration of a model phosphotransfer transition state complex.
The phosphoenolpyruvate:glycose transferase system (PTS) is a prototypic signaling system responsible for the vectorial uptake and phosphorylation of carbohydrate substrates. The accompanying papers describe the proteins and product of the Escherichia coli N, N-diacetylchitobiose ((GlcNAc)(2)) PTS-mediated permease. Unlike most PTS transporters, the Chb system is composed of two soluble proteins, IIA(Chb) and IIB(Chb), and one transmembrane receptor (IIC(Chb)). The oligomeric states of PTS permease proteins and phosphoproteins have been difficult to determine. Using analytical ultracentrifugation, both dephospho and phosphorylated IIA(Chb) are shown to exist as stable dimers, whereas IIB(Chb), phospho-IIB(Chb) and the mutant Cys10SerIIB(Chb) are monomers. The mutant protein Cys10SerIIB(Chb) is unable to accept phosphate from phospho-IIA(Chb) but forms a stable higher order complex with phospho-IIA(Chb) (but not with dephospho-IIA(Chb)). The stoichiometry of proteins in the purified complex was determined to be 1:1, indicating that two molecules of Cys10SerIIB(Chb) are associated with one phospho-IIA(Chb) dimer in the complex. The complex appears to be a transition state analogue in the phosphotransfer reaction between the proteins. A model is presented that describes the concerted assembly and disassembly of IIA(Chb)-IIB(Chb) complexes contingent on phosphorylation-dependent conformational changes, especially of IIA(Chb).
Keyhani
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Department of Biology and McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Rodgers
M E
ME
Demeler
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Hansen
J C
JC
Roseman
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S
D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D004187:Disaccharides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D002352:Carrier Proteins; D004187:Disaccharides; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C010913:N,N-diacetylchitobiose; D010770:Phosphotransferases