PMID:8969208
| Citation |
Pelton, JG, Torchia, DA, Remington, SJ, Murphy, KP, Meadow, ND and Roseman, S (1996) Structures of active site histidine mutants of IIIGlc, a major signal-transducing protein in Escherichia coli. Effects on the mechanisms of regulation and phosphoryl transfer. J. Biol. Chem. 271:33446-56 |
|---|---|
| Abstract |
IIIGlc (also called IIAGlc), a major signal-transducing protein in Escherichia coli, is also a phosphorylcarrier in glucose uptake. The crystal and NMR structures of IIIGlc show that His90, the phosphoryl acceptor, adjoins His75 in the active site. Glutamine was substituted for His-, giving H75QIIIGlc and H90QIIIGlc, respectively (Presper, K. A., Wong, C.-Y., Liu, L., Meadow, N. D., and Roseman, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4052-4055), but the mutants showed unexpected properties. H90QIIIGlc loses regulatory functions of IIIGlc, and the phosphoryltransfer rates between HPr/H75QIIIGlc are 200-fold less than HPr/IIIGlc (Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33440-33445). X-ray crystallography, differential scanning calorimetry, and NMR have now been used to determine the structures of the mutants (phospho-H75QIIIGlc was studied by NMR). The three methods gave completely consistent results. Except for the His to Gln substitutions, the only significant structural changes were in a few hydrogen bonds. H90QIIIGlc contains two structured water molecules (to Gln90), which could explain its inability to regulate glycerol kinase. Phospho-IIIGlc contains a chymotrypsin-like, hydrogen bond network (Thr73-His75-O--phosphoryl), whereas phospho-H75QIIIGlc contains only one bond (Gln75-O--phosphoryl). Hydrogen bonds play an essential role in a proposed mechanism for the phosphoryltransfer reaction. |
| Links | |
| Keywords |
Binding Sites; Calorimetry; Crystallography, X-Ray; Escherichia coli/chemistry; Escherichia coli Proteins; Histidine; Hydrogen Bonding; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Models, Molecular; Mutagenesis, Site-Directed; Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry; Phosphorylation |
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