The transport/phosphorylation of N,N'-diacetylchitobiose in Escherichia coli. Characterization of phospho-IIB(Chb) and of a potential transition state analogue in the phosphotransfer reaction between the proteins IIA(Chb) AND IIB(Chb).
Enzyme II permeases of the phosphoenolpyruvate:glycose phosphotransferase system comprise one to five separately encoded polypeptides, but most contain similar domains (IIA, IIB, and IIC). The phosphoryl group is transferred from one domain to another, with histidine as the phosphoryl acceptor in IIA and cysteine as the acceptor in certain IIB domains. IIB(Chb) is a phosphocarrier in the uptake/phosphorylation of the chitin disaccharide, (GlcNAc)(2) by Escherichia coli and is unusual because it is separately encoded and soluble. Both the crystal and solution structures of a IIB(Chb) mutant (C10S) have been reported. In the present studies, homogeneous phospho-IIB(Chb) was isolated, and the phosphoryl-Cys linkage was established by (31)P NMR spectroscopy. Rate constants for the hydrolysis of phospho-IIB(Chb) plotted versus pH gave the same shape peak reported for the model compound, butyl thiophosphate, but was shifted about 4 pH units. Evidence is presented for a stable complex between homogeneous Cys10SerIIB(Chb) (which cannot be phosphorylated) and phospho-IIA(Chb), but not with IIA(Chb). The complex (a tetramer (3)) contains equimolar quantities of the two proteins and has been chemically cross-linked. It appears to be an analogue of the transition state complex in the reaction: phospho-IIA(Chb) + IIB(Chb) <--> IIA(Chb) + phospho-IIB(Chb). This is apparently the first report of the isolation of a transition state analogue in a protein-protein phosphotransfer reaction.
Keyhani
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Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Bacia
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Roseman
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D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004187:Disaccharides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D016366:Open Reading Frames; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004187:Disaccharides; D011994:Recombinant Proteins; C010913:N,N-diacetylchitobiose