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.
Academic Article
Overview
abstract
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.
