Isolation and characterization of IIAChb, a soluble protein of the enzyme II complex required for the transport/phosphorylation of N, N'-diacetylchitobiose in Escherichia coli.
N,N'-Diacetylchitobiose is transported/phosphorylated in Escherichia coli by the (GlcNAc)(2)-specific Enzyme II permease of the phosphoenolpyruvate:glycose phosphotransferase system. IIA(Chb), one protein of the Enzyme II complex, was cloned and purified to homogeneity. IIA(Chb) and phospho-IIA(Chb) form stable homodimers (). Phospho-IIA(Chb) behaves as a typical epsilon2-N (i.e. N-3) phospho-His protein. However, the rate constants for hydrolysis of phospho-IIA(Chb) at pH 8.0 unexpectedly increased 7-fold between 25 and 37 degrees C and increased approximately 4-fold with decreasing protein concentration at 37 degrees C (but not 25 degrees C). The data were explained by thermal denaturation studies using CD spectroscopy. IIA(Chb) and phospho-IIA(Chb) exhibit virtually identical spectra at 25 degrees C (approximately 80% alpha-helix), but phospho-IIA(Chb) loses about 30% of its helicity at 37 degrees C, whereas IIA(Chb) shows only a slight change. Furthermore, the T(m) for thermal denaturation of IIA(Chb) was 54 degrees C, only slightly affected by concentration, whereas the T(m) for phospho-IIA(Chb) was much lower, ranging from 40 to 46 degrees C, depending on concentration. In addition, divalent cations (Mg(2+), Cu(2+), and Ni(2+)) have a dramatic and differential effect on the structure, depending on the state of phosphorylation of the protein. Thus, phosphorylation destabilizes IIA(Chb) at 37 degrees C, potentially affecting the monomer/dimer transition, which correlates with its chemical instability at this temperature. The physiological consequences of this phenomenon are briefly considered.
Keyhani
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Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Boudker
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Roseman
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D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D019281:Dimerization; D004187:Disaccharides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics
D002352:Carrier Proteins; D004187:Disaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C010913:N,N-diacetylchitobiose