Norman D. Meadow

Understanding glucose transport by the bacterial phosphoenolpyruvate:glycose phosphotransferase system on the basis of kinetic measurements in vitro.

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 theK 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.

Modified assay procedures for the phosphotransferase system in enteric bacteria.

Abstract Conditions for the assay of individual components of the bacterial phosphotransferase system (PTS) are presented wich offer two important improvements over earlier methods. First, a lactate dehydrogenase-coupled assay for phosphocarrier proteins (HPr, FPr, and Factor IIIGle) which permits their measurement in either pure or partially pure form was developed. Quantitation by this assay does not rely on the level of activity of the enzymes used. Second, conditions under which Enzyme I activity was proportional to enzyme concentration are given. With these methods levels of PTS components have been measured that are 2-to 20-fold higher than those previously reported. These levels can now account for various PTS functions measured in vivo. Further, we have shown that the phosphocarrier proteins HPr and Factor IIIGle are substrates for their respective enzymes which show typical Michaelis-Menten kineties. In addition, a method for the partial purification of Enzyme II-BGle essentially free of Enzyme IIMan activity is presented.

Rate and Equilibrium Constants for Phosphoryltransfer between Active Site Histidines of Escherichia coli HPr and the Signal Transducing Protein IIIGlc

The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) plays a central role in catabolizing many sugars; regulation is effected by phosphorylation of PTS proteins. In Escherichia coli, the phosphoryltransfer sequence for glucose uptake is: PEP → Enzyme I(His191) → HPr(His15) → IIIGlc(His90) → IIGlc(Cys421) → glucose. A rapid quench method has now been developed for determining the rate and equilibrium constants of these reactions. The method was validated by control experiments, and gave the following results for phosphoryltransfer between the following protein pairs. For phospho-HPr/IIIGlc (and HPr/phospho-IIIGlc), k1 = 6.1 × 107 M−1 s−1, k−1 = 4.7 × 107; for the mutant H75QIIIGlc in place of IIIGlc, k1 = 2.8 × 105 M−1 s−1, k−1 = 2.3 × 105. The derived Keq values agreed with the Keq obtained without use of the rapid quench apparatus. Keq for both reactions is 1-1.5. The rate of phosphoryltransfer between HPr and wild type IIIGlc is close to a diffusion-controlled process, while the reactions involving the mutant H75QIIIGlc are 200-fold slower. These rate differences are explained by an hypothesis for the mechanism of phosphoryltransfer between HPr and IIIGlc based on the structures of mutant and wild type proteins (see Pelton et al. (Pelton, J. G., Torchia, D. A., Remington, S. J., Murphy, K. P., Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33446-33456)).

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

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.

Transient State Kinetics of Enzyme IICBGlc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli EQUILIBRIUM AND SECOND ORDER RATE CONSTANTS FOR THE GLUCOSE BINDING AND PHOSPHOTRANSFER REACTIONS

During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIAGlc and Enzyme IICBGlc, the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system. Transient state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIAGlc to IICBGlc to Glc) and also the second order rate constants for the transfer from phospho-IIAGlc to molecularly cloned IIBGlc, the soluble, cytoplasmic domain of IICBGlc. The rate constants for the forward and reverse phosphotransfer reactions between IIAGlc and IICBGlc were 3.9 × 106 and 0.31 × 106 m–1 s–1, respectively, and the rate constant for the physiologically irreversible reaction between [P]IICBGlc and Glc was 3.2 × 106 m–1 s–1. From the rate constants, the equilibrium constants for the transfer of the phospho-group from His90 of [P]IIAGlc to the phosphorylation site Cys of IIBGlc or IICBGlc were found to be 3.5 and 12, respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl phosphotransferase system proteins. In these studies, preparations of IICBGlc were invariably found to contain endogenous, firmly bound Glc (estimated K′D ∼10–7 m). The bound Glc was kinetically competent and was rapidly phosphorylated, indicating that IICBGlc has a random order, Bi Bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.

Effects of Mutations and Truncations on the Kinetic Behavior of IIAGlc, a Phosphocarrier and Regulatory Protein of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

IIAGlc, a component of the glucose-specific phosphoenolpyruvate:phosphotransferase system (PTS) of Escherichia coli, is important in regulating carbohydrate metabolism. In Glc uptake, the phosphotransfer sequence is: phosphoenolpyruvate → Enzyme I → HPr → IIAGlc → IICBGlc → Glc. (HPr is the first phosphocarrier protein of the PTS.) We previously reported two classes of IIAGlc mutations that substantially decrease the P-transfer rate constants to/from IIAGlc. A mutant of His75 which adjoins the active site (His90), (H75Q), was 0.5% as active as wild-type IIAGlc in the reversible P-transfer to HPr. Two possible explanations were offered for this result: (a) the imidazole ring of His75 is required for charge delocalization and (b) H75Q disrupts the hydrogen bond network: Thr73, His75, phospho-His90. The present studies directly test the H-bond network hypothesis. Thr73 was replaced by Ser, Ala, or Val to eliminate the network. Because the rate constants for phosphotransfer to/from HPr were largely unaffected, we conclude that the H-bond network hypothesis is not correct. In the second class of mutants, proteolytic truncation of seven residues of the IIAGlc N terminus caused a 20-fold reduction in phosphotransfer to membrane-bound IICBGlc from Salmonella typhimurium. Here, we report the phosphotransfer rates between two genetically constructed N-terminal truncations of IIAGlc (Δ7 and Δ16) and the proteins IICBGlc and IIBGlc (the soluble cytoplasmic domain of IICBGlc). The truncations did not significantly affect reversible P-transfer to IIBGlc but substantially decreased the rate constants to IICBGlc in E. coli and S. typhimurium membranes. The results support the hypothesis (Wang, G., Peterkofsky, A., and Clore, G. M. (2000) J. Biol. Chem. 275, 39811–39814) that the N-terminal 18-residue domain “docks” IIAGlc to the lipid bilayer of membranes containing IICBGlc.

[70] Assays for the phosphotransferase system from Salmonella typhimurium

Publisher Summary This chapter describes the procedures for the preparation of membranes and for the partial purification of Enzyme II-B Glc , which, as sources of Enzyme II, are required for the sugar phosphorylation assays. Sugar phosphorylation assay is based on the separation, by ion-exchange chromatography, of radioactively labeled unreacted sugar from radioactive sugar phosphate formed by the complete phosphotransferase system. In spectrophotometric assay, the pyruvate formed during the phosphorylation of the protein is measured by following the oxidation of NADH by lactate dehydrogenase as a function of time, using a recording spectrophotometer at 340 nm with a temperature-regulated water jacket (30 ° C) surrounding the cuvette holder. The assay procedure for preparation of the membrane components of the PTS involves preparation of membranes and the preparation of II-B Glc . Although the activity of Enzyme II-B Glc in membranes from SB1687 is relatively high compared to the activity of Enzyme II-B Man , the latter enzyme shows some background activity in the sugar phosphorylation assay.

[69] General description and assay principles

Publisher Summary This chapter presents the general description of phosphotransferase system (PTS) and its assay principles. PTS is widely distributed in obligate and facultative anaerobic bacteria and has occasionally been found in strict aerobes. The overall reaction catalyzed by the PTS is the transfer of the phosphoryl group from phosphoenolpyruvate to the sugar substrate, yielding the corresponding sugar-6-P, with the exception of fructose, which is phosphorylated by an inducible fructose-specific PTS at the C-1 position or, less effectively, by a constitutive PTS at C-6. Two types of assays are used to measure the components of the phosphotransferase system—the spectrophotometric assay and the sugar phosphorylation assay. The spectrophotometric assay is based on determining the stoichiometry of phosphate incorporation into certain PTS proteins; the quantity of the given PTS protein in the sample can be determined by measuring pyruvate formation resulting from phosphate transfer from phosphoenolpyruvate to the unknown. The sugar phosphorylation assay is based on the separation of radioactively labeled sugar phosphate from unreacted radioactive sugar.

[73] IIIGle from Salmonella typhimurium

Publisher Summary This chapter discusses two purification procedures and the properties of the enzyme III Glc from Salmonella typhimurium . III Glc can be purified by two procedures; pure protein is first produced by procedure A and this protein can then be used to obtain antibodies. Once the antibodies are available, the simpler and much more rapid procedure B is used. The purification of III Glc is monitored with anti-III Glc antiserum using rocket immunoelectrophoresis and related techniques. The sugar phosphorylation assay is used to confirm the results obtained by immunoelectrophoresis methods. Procedure A involves preparation of crude extract, precipitation with protamine sulfate, diethylaminoethyl (DEAE)-cellulose chromatography, Sephadex G-75 column chromatography, preparative isoelectric focusing, and preparative polyacrylamide gel electrophoresis. Procedure B involves adsorption and elution from antibody column, Sephadex G-75 column chromatography, and preparative polyacrylamide gel electrophoresis.