STRUCTURES OF ACTIVE-SITE HISTIDINE MUTANTS OF IIIGLC, A MAJOR SIGNAL-TRANSDUCING PROTEIN IN ESCHERICHIA-COLI - EFFECTS ON THE MECHANISM OFREGULATION AND PHOSPHORYL TRANSFER

IIIGlc (also called IIA(Glc)), a major signal-transducing protein in E scherichia coli, is also a phosphorylcarrier in glucose uptake, The cr ystal and NMR structures of IIIGlc show that His(90), the phosphoryl a cceptor, adjoins His(75) in the active site, Glutamine was substituted for His-, giving (IIIGlc)-I-H75Q and (IIIGlc)-I-H90Q, respectively (P resper, K. A., Wong, C.-Y., Liu, L., Meadow, N. D., and Roseman, S. (1 989) Proc, Natl. Acad. Sci. U.S.A. 86, 4052-4055), but the mutants sho wed unexpected properties, (IIIGlc)-I-H90Q loses regulatory functions of IIIGlc, and the phosphoryltransfer rates between Hpr/(IIIGlc)-I-H75 Q are 200-fold less than HPr/IIIGlc (Meadow, N. D., and Roseman, S. (1 996) J. Biol. Chem. 271, 33440-33445), X-ray crystallography, differen tial scanning calorimetry, and NMR have now been used to determine the structures of the mutants (phospho-(IIIGlc)-I-H75Q was studied by NMR ), The three methods gave completely consistent results. Except for th e His to Gin substitutions, the only significant structural changes we re in a few hydrogen bonds, (IIIGlc)-I-H90Q contains two structured wa ter molecules (to Gln(90)), which could explain its inability to regul ate glycerol kinase. Phospho-IIIGlc contains a chymotrypsin like, hydr ogen bond network (Thr(73)-His(75) -O(-)phosphoryl), whereas phospho-( IIIGlc)-I-H75Q contains only one bond (Gln(75)-O--phosphoryl), Hydroge n bonds play an essential role in a proposed mechanism for the phospho ryltransfer reaction.