IIIG'C is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoeno1pyruvate:glycose phosphotransferase system from Escherichia coli. The 'H, "N, and I3C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGLC have been assigned using two-dimensional IH-"N and 'H-I3C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional I3C-I3C-'H correlation spectroscopy via J,, coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly '5N/'3C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The "N and 13C chemical shifts were used t o determine that His-75 exists predominantly in the NE'-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIG'C, and that His-90 exists primarily in the N6'-H state in the unphosphorylated protein. Upon phosphorylation of the NE* nitrogen of His-90, the N*' nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 'H, "N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK, values for both His-75 and His-90 in IIIG'' and His-75 in phospho-IIIG'C are less than 5.0, and that the pK, value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIG", and implications for proposed mechanisms of phosphoryl transfer are discussed.

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The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected. To address the issue of whether the behavior in vivo of the PTS can be understood in terms of these enzyme kinetics, a detailed kinetic model was constructed. Each overall phosphotransfer reaction was separated into two elementary reactions, the first entailing association of the phosphoryl donor and acceptor into a complex and the second entailing dissociation of the complex into dephosphorylated donor and phosphorylated acceptor. Literature data on the K m values and association constants of PTS proteins for their substrates, as well as equilibrium and rate constants for the overall phosphotransfer reactions, were related to the rate constants of the elementary steps in a set of equations; the rate constants could be calculated by solving these equations simultaneously. No kinetic parameters were fitted. As calculated by the model, the kinetic parameter values in vitro could describe experimental results in vivo when varying each of the PTS protein concentrations individually while keeping the other protein concentrations constant. Using the same kinetic constants, but adjusting the protein concentrations in the model to those present in cell-free extracts, the model could reproduce experiments in vitro analyzing the dependence of the flux on the total PTS protein concentration. For modeling conditions in vivo it was crucial that the PTS protein concentrations be implemented at their high in vivo values. The model suggests a new interpretation of results hitherto not understood; in vivo, the major fraction of the PTS proteins may exist as complexes with other PTS proteins or boundary metabolites, whereas in vitro, the fraction of complexed proteins is much smaller.In many bacteria, the phosphoenolpyruvate:glycose phosphotransferase system (PTS) 1 is involved in the uptake and concomitant phosphorylation of a variety of carbohydrates (for reviews, see Refs. 1 and 2). The PTS is a group transfer pathway; a phosphoryl group derived from phosphoenolpyruvate (PEP) is transferred sequentially along a series of proteins to the carbohydrate molecule. The sequence of phosphotransfer is from PEP to the general cytoplasmic PTS proteins enzyme I (EI) and HPr and, in the case of glucose, further to the carbohydrate-specific cytoplasmic IIA Glc , membrane-bound IICB Glc (the glucose permease), and glucose. For other carbohydrates, specific enzymes II exist (with the A, B, and C domains present either as a single polypeptide or as multiple proteins, depending on the carbohydrate that is transported), which accept the phosphoryl group from HPr (1-4). Apart from its direct role in the above phosphotransfer and its indirect role in transport, IIA Glc is an important signaling molecule, mediating catabolite repression (reviewed in Refs. 1, 2, and 5). The presence or absence of a PTS substrate affects the IIA Glc phosphorylation state; in the absence of PTS substrate, phosphory...