PubMed Health⌕ Search

Biomedical subjects

E B Waygood

Publications and source records attributed to E B Waygood.

At least 55 records · Page 3Linked to original sources
PubMed1986

Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system has two sites of phosphorylation per dimer.

Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli has been reported to contain one phosphorylation site per dimer and thus operates by either a half of the sites or a flip-flop mechanism [Misset, O., & Robillard, G. T. (1982) Biochemistry 21, 3136-3142; Hoving, T., ten Hoeve-Duurkens, R., & Robillard, G. T. (1984) Biochemistry 23, 4335-4340]. In this paper, the determination of two phosphorylation sites per dimer of enzyme I was made by using a number of different methods. In some experiments, less than two sites per dimer were found, but a concomitant loss in enzyme I activity was also found. The phosphorylated residue in enzyme I was shown to have the properties expected for a N3-phosphohistidinyl residue.

Buffers↗
PubMed1986

The bacterial phosphotransferase system: kinetic characterization of the glucose, mannitol, glucitol, and N-acetylglucosamine systems.

The kinetic mechanisms by which the glucose, glucitol, N-acetylglucosamine, and mannitol enzymes II catalyze sugar phosphorylation have been investigated in vitro. Lineweaver-Burk analyses indicate that the glucose and glucitol enzymes II catalyze sugar phosphorylation by a sequential mechanism when the two substrates are phospho-enzyme III and sugar. The N-acetylglucosamine and mannitol enzymes II, which do not function with an enzyme III, catalyze sugar phosphorylation by a ping-pong mechanism when the two substrates are phospho-HPr and sugar. These results, as well as previously published kinetic characterizations, suggest a common kinetic mechanism for all enzymes II of the system. It is suggested that all enzymes II and enzyme II-III pairs arose from a single (fused) gene product containing two sites of phosphorylation and that phosphoryl transfer from the second phosphorylation site to sugar can only occur when the enzyme II-III pair is present in the associated state.

Acetylglucosamine↗
PubMed1986

Phosphoproteins and the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus salivarius. Detection of two different ATP-dependent phosphorylations of the phosphocarrier protein HPr.

Phosphoproteins which arise from incubation of Streptococcus salivarius ATCC25975 crude extracts with [32P]phosphoenolpyruvate and [gamma-32P]ATP, were separated and detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. These procedures were carried out using the methodology that has been developed to allow for the detection of phosphoproteins containing 1-P-histidinyl and 3-P-histidinyl residues, and also to distinguish between these and phosphoproteins containing acid-stable phosphoamino acids such as phosphoserine, phosphothreonine, and phosphotyrosine. Extracts of cells which had been grown with various sugars as carbon sources were investigated to determine both constitutive and inducible phosphoproteins. No evidence was found for phosphoproteins specifically induced by a sugar, and in particular no evidence was found for any IIIsugar phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Incubation with [gamma-32P]ATP showed that histidine-containing phosphocarrier protein (HPr) of the PTS could be phosphorylated to give both acid-stable and acid-labile phosphoamino acid residues. The acid-labile ATP-dependent phosphorylation activity was activated by glucose-6-P and appeared to produce a 3-P-histidinyl residue in HPr.

Adenosine Triphosphate↗
PubMed1985

Characterization of phosphorylated histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

The histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate:sugar phosphotransferase system, when phosphorylated, contains a 1-phosphohistidinyl (1-P-histidinyl) residue (His-15). The properties of this 1-P-histidinyl residue were investigated by using phospho-HPr (P-HPr), P-HPr-1, and P-HPr-2. HPr-1 and HPr-2 are deamidated forms of HPr produced by boiling. In addition, HPr-1 produced during frozen storage was investigated. Both pH and temperature dependencies of the rate of hydrolysis of the phosphoryl group of the 1-P-histidinyl residue were investigated. The results show that the 1-P-histidinyl residue in HPr and HPr-1 has significantly different properties from free 1-P-histidine and that these differences are attributable to the active-site residues Glu-66 and Arg-17 and the pK of the imidazole group of the 1-P-histidinyl residue in P-HPr. The 1-P-histidinyl residue in P-HPr and P-HPr-1 shows a greater lability at physiological pH than the free amino acid. A proposal for the active site of P-HPr is made on the basis of these results and the recently obtained tertiary structure. In contrast, the hydrolysis properties of the 1-P-histidinyl residue in P-HPr-2 were similar to those obtained for either free 1-P-histidine or denatured P-HPr. The loss of activity that is associated with boiling HPr was shown to be due to HPr-2 formation as HPr-1 was found to be fully active.

Bacterial Proteins↗
PubMed1985

Bacterial phosphotransferase system: regulation of the glucose and mannose enzymes II by sulfhydryl oxidation.

We have investigated the effect of oxidizing agents on methyl alpha-glucoside phosphorylation by the Escherichia coli phosphotransferase system (PTS). Oxidizing agents inhibited methyl alpha-glucoside phosphorylation at low methyl alpha-glucoside concentrations, and the degree of inhibition was shown to decrease with increasing concentrations of methyl alpha-glucoside. Results of studies with mutant bacteria and substrate analogues of the glucose and mannose enzymes II showed that contrary to the interpretation of Robillard and Konings [Robillard, G. T., & Konings, W. N. (1981) Biochemistry 20, 5025-5032] the apparent change in the Km value for methyl alpha-glucoside phosphorylation induced by sulfhydryl oxidation is not due to the formation of a low-affinity, oxidized form of the glucose enzyme II. Rather, the results are explained by the presence of two phosphotransferase systems that phosphorylate methyl alpha-glucoside with different affinities and that are differentially sensitive to oxidizing agents. The low Km system corresponds to the glucose enzyme II, which is strongly inhibited by potassium ferricyanide, phenazine methosulfate, and plumbagin. The high Km system corresponds to the mannose enzyme II, which is less sensitive to inhibition by these oxidizing agents. This differential sensitivity to inhibition by oxidizing agents can account for the apparent Km change for methyl alpha-glucoside phosphorylation reported by Robillard and Konings. The physiological significance of sulfhydryl oxidation in the enzymes II of the PTS has yet to be ascertained.

Deoxyglucose↗
PubMed1985

Bacterial phosphotransferase system: regulation of mannitol enzyme II activity by sulfhydryl oxidation.

The mechanism by which the oxidation-reduction potential regulates the bacterial phosphotransferase system in Escherichia coli has been investigated. Transphosphorylation experiments verified that the oxidizing agent, potassium ferricyanide, directly inhibits mannitol enzyme II activity. Phosphorylation of enzyme IImtl with enzyme I, heat-stable phosphocarrier protein of the phosphotransferase system, and phosphoenolpyruvate partially protects the enzyme from ferricyanide inhibition. The enzyme is even less sensitive to inhibition during catalytic turnover. Preincubation of unphosphorylated enzyme with ferricyanide, however, reversibly inactivates it even at high mannitol concentrations. The results are inconsistent with a regulatory mechanism in which sulfhydryl oxidation influences the affinity of the enzyme for the substrate. Instead, it is concluded that the oxidized enzyme is inactive.

Escherichia coli↗
PubMed1985

Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) found in enteric bacteria is a complex enzyme system consisting of a non-sugar-specific phosphotransfer protein called Enzyme I, two small non-sugar-specific phosphocarrier substrates of Enzyme I, designated HPr and FPr, and at least 11 sugar-specific Enzymes II or Enzyme II-III pairs which are phosphorylated at the expense of phospho-HPr or phospho-FPr. In this communication, evidence is presented which suggests that these proteins share a common evolutionary origin and that a fructose-specific phosphotransferase may have been the primordial ancestor of them all. The evidence results from an evaluation of 1) PTS protein sequence data; 2) structural analysis of operons encoding proteins of the PTS; 3) genetic regulatory mechanisms controlling expression of these operons; 4) enzymatic characteristics of the PTS systems; 5) immunological cross reactivities of these proteins; 6) comparative studies of phosphotransferase systems from evolutionarily divergent bacteria; 7) the nature of the phosphorylated protein intermediates; 8) molecular weight comparisons among the different Enzymes II and Enzyme II-III pairs; and 9) interaction studies involving different PTS protein constituents. The evidence leads to a unifying theory concerning the evolutionary origin of the system, explains many structural, functional, and regulatory properties of the phosphotransferase system, and leads to specific predictions which should guide future research concerned with genetic, biochemical, and physiological aspects of the system.

Biological Evolution↗
PubMed1985

Allosteric regulation of glycerol kinase by enzyme IIIglc of the phosphotransferase system in Escherichia coli and Salmonella typhimurium.

The mechanism by which enzyme IIIglc of the bacterial phosphotransferase system regulates the activity of crystalline glycerol kinase from Escherichia coli has been studied, and the inhibitory effects have been compared with those produced by fructose-1,6-diphosphate. It was shown that the free, but not the phosphorylated, form of enzyme IIIglc inhibits the kinase. Mutants of Salmonella typhimurium were isolated which were resistant to inhibition by either enzyme IIIglc (glpKr mutants) or fructose-1,6-diphosphate (glpKi mutants), and each mutant type was shown to retain full sensitivity to inhibition by the other regulatory agent. Other mutants were fully or partially resistant to regulation by both agents. The two regulatory sites on the kinase are evidently distinct but must overlap or interact functionally. Kinetic analyses have revealed several mechanistic features of the regulatory interactions. (i) Inhibition by both allosteric regulatory agents is strongly pH dependent, with maximal inhibition occurring at ca. pH 6.5 under the assay conditions employed. (ii) Binding of enzyme IIIglc to glycerol kinase is also pH dependent, the Ki being near 4 microM at pH 6.0 but near 10 microM at pH 7.0. (iii) Whereas fructose-1,6-diphosphate inhibition apparently requires that the enzyme exist in a tetrameric state, both the dimer and the tetramer appear to be fully sensitive to enzyme IIIglc inhibition. (iv) Inhibition by enzyme IIIglc (like that by fructose-1,6-diphosphate) is noncompetitive with respect to both substrates. (v) The inhibitory responses of glycerol kinase to fructose-1, 6-diphosphate and enzyme IIIglc show features characteristic of positive cooperativity at low inhibitor concentration. (vi) Neither agent inhibits completely at high inhibitor concentration. (vii) Apparent negative cooperativity with respect to ATP binding is observed with purified E. coli glycerol kinase, with glycerol kinase in crude extracts of wild-type S. typhimurium cells, and with glpKr and glpKi mutant forms of glycerol kinase from S. typhimurium. These results serve to characterize the regulatory interactions which control the activity of glycerol kinase by fructose-1,6-diphosphate and by enzyme IIIglc of the phosphotransferase system.

Allosteric Regulation↗
PubMed1985

Evidence for covalently cross-linked dimers and trimers of enzyme I of the Escherichia coli phosphotransferase system.

Enzyme I of the bacterial phosphotransferase system catalyzes transfer of the phosphoryl moiety from phosphoenolpyruvate to both of the heat-stable phosphoryl carrier proteins of the phosphotransferase system, HPr and FPr. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-pressure liquid chromatography, we demonstrated the existence of covalently cross-linked enzyme I dimers and trimers. Enzyme I exchange assays and phosphorylation experiments with [32P]phosphoenolpyruvate showed that covalent dimers and trimers are catalytically active. Inhibitors of the enzyme I-catalyzed phosphoenolpyruvate-pyruvate exchange block the phosphorylation of enzyme I dimers and trimers. Inhibition of the activity of enzyme I by N-ethylmaleimide, but not that by p-chloromercuriphenylsulfonate, could be overcome by high concentrations of enzyme, suggesting that N-ethylmaleimide modification changes the associative properties of enzyme I. We present evidence for two distinct classes of sulfhydryl groups in enzyme I.

Chromatography, High Pressure Liquid↗
PubMed1984

Isoelectrophoretic separation and the detection of soluble proteins containing acid-labile phosphate: use of the phosphoenolpyruvate:sugar phosphotransferase system as a model system for N1-P-histidine- and N3-P-histidine-containing proteins.

Procedures have been developed for the detection of acid-labile phosphorylations of proteins. The phosphoproteins were separated by native isoelectric focusing while maintaining the gel at about 0 degree C, and denaturing urea-Nonidet isoelectric focusing gels were adapted to run at -10 degrees C. The proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), HPr, which contains 1-P-histidine, and factor IIIglc and enzyme I, which contain 3-P-histidine when they are phosphorylated, were used to develop the conditions. Autoradiography of [32P]-labeled phosphoproteins was carried out on frozen gels which had not been acid fixed in order to avoid hydrolysis of the phosphohistidines . The frozen gels were subsequently fixed and stained, and reautoradiography revealed whether the phosphoproteins were acid stable or labile. In addition to the known proteins of the PTS, at least one other protein whose phosphorylation was dependent on enzyme I and HPr was found in Salmonella typhimurium and Escherichia coli [E.B. Waygood , and R.L. Mattoo (1983) Canad . J. Biochem. Cell Biol. 61, 150-153]. Initial experiments with rat tissues have demonstrated acid-labile phosphorylations in proteins which were either [gamma-32P]ATP or [32P]phosphoenolpyruvate dependent. The interconversion of phosphoenolpyruvate and ATP in crude extracts of bacterial cells was examined, and appropriate controls were found. Protein phosphorylation dependent upon phosphoenolpyruvate was much greater in S. typhimurium and E. coli than the corresponding ATP-dependent phosphorylation, while the opposite was found for rat tissues.

Adenosine Triphosphate↗
PubMed1984

Activation of phosphoenolpyruvate-dependent protein kinase by cytidine 5'-triphosphate in rat skeletal muscle.

The effects of nucleotides on phosphoenolpyruvate-dependent protein kinase in rat skeletal muscle were examined. Various ribonucleotide triphosphates were tested and the protein kinase reaction was maximally activated by cytidine 5'-triphosphate followed by uridine 5'-triphosphate and guanosine 5'-triphosphate. No activation by adenosine 5'-triphosphate was observed. Cytidine 5'-diphosphate also activated the reaction but to a significantly lesser extent. Cytidine 5'-monophosphate and cyclic cytidine 3',5'-monophosphate showed no effect whereas cytidine was slightly inhibitory.

Adenosine Triphosphate↗
PubMed1984

Phosphoproteins and the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium and Escherichia coli: evidence for IIImannose, IIIfructose, IIIglucitol, and the phosphorylation of enzyme IImannitol and enzyme IIN-acetylglucosamine.

Phosphoproteins produced by the incubation of crude extracts of Salmonella typhimurium and Escherichia coli with either [32P]phosphoenolpyruvate or [gamma 32P]ATP have been resolved and detected using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Simple techniques were found such that distinctions could be made between phosphoproteins containing acid-labile or stable phosphoamino acids and between N1-P-histidine and N3-P-histidine. Phosphoproteins were found to be primarily formed from phosphoenolpyruvate, but because of an efficient phosphoexchange, ATP also led to the formation of the major phosphoenolpyruvate-dependent phosphoproteins. These proteins had the following apparent subunit molecular weights: 65,000, 65,000, 62,000, 48,000, 40,000, 33,000, 25,000, 20,000, 14,000, 13,000, 9,000, 8,000. Major ATP-dependent phosphoproteins were detected with apparent subunit molecular weights of 75,000, 46,000, 30,000, and 15,000. Other minor phosphoproteins were detected. The phosphorylation of the 48,000- and 25,000-MW proteins by phosphoenolpyruvate was independent of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS phosphoproteins were identified as enzyme I (soluble; MW = 65,000); enzyme IIN-acetylglucosamine (membrane bound; MW = 65,000); enzyme IImannitol (membrane bound; MW = 62,000); IIIfructose (soluble; MW = 40,000); IIImannose (partially membrane associated; MW = 33,000); IIIglucose (soluble; MW = 20,000); IIIglucitol (soluble; MW = 13-14,000); HPr (soluble; MW = 9,000); FPr (fructose induced HPr-like protein (soluble; MW = 8,000). HPr and FPr are phosphorylated on the N-1 position of a histidyl residue while all the others appear to be phosphorylated on an N-3 position of a histidyl residue. These studies identify some previously unknown proteins of the PTS and show the phosphorylation of others, which although previously known, had not been shown to be phosphoproteins.

Acetylglucosamine↗
PubMed1983

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle.

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.

Adenosine Triphosphate↗
PubMed1983

Determination of the levels of HPr and enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium.

The levels of histidine-containing protein HPr and enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system of Escherichia coli strains 1100, NC3, W3110, and P650 and Salmonella typhimurium strains SB3507 and LJ144 have been determined by quantitative sugar phosphorylation assay and immunochemically. The levels have been determined for cells grown on minimal salts with glucose, fructose, mannitol, glycerol, and lactate and on nutrient broth. All determinations indicate a two- to three-fold change in the levels of enzyme I and HPr between growth on hexoses, which gave the higher levels, and the other growth substrates. The highest levels were not always found in glucose-grown cells. Antibodies were produced in rabbits using purified proteins from E. coli P650. The activity measurements and immunochemically determined enzyme I protein gave specific activities in the crude extracts of E. coli strains which were similar to that of the pure enzyme. The wild-type S. typhimurium enzyme I in crude extracts did not have the same immunochemical reactivity, although there was a considerable cross-reaction and the specific activity appeared to be half that of pure enzyme I. The HPr from both E. coli and S. typhimurium behaved identically and, although the immunoprecipitation was weak, it did indicate that HPr assays may not be as reliable as the enzyme I assays. The relative amounts of enzyme I and HPr found indicate that there are between 10- and 20-fold more HPr molecules in a cell than enzyme I subunits which form active dimers.

Animals↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. The glucose receptors of the Salmonella typhimurium phosphotransferase system.

We have previously reported that glucose can be phosphorylated by phospho-HPr and two sugar-specific pairs of proteins of the Escherichia coli and Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Each of the sugar-specific complexes comprises two proteins, lipid, and divalent cation, and each is present in membranes isolated from wild type cells. For reasons described in this report, one of the complexes is designated IIGlc and the other IIMan. The IIMan complex has previously been separated into its protein components, II-A and II-B (Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 1407-1418), while the accompanying reports describe dissociation of the IIGlc complex into its components, IIIGlc and II-BGlc. Curtis and Epstein (Curtis, S. J., and Epstein, W. (1975) J. Bacteriol. 122, 1189-1199) first showed that there are two phosphotransferase systems in whole cells responsible for glucose uptake and obtained the respective mutants, now designated ptsG and ptsM. The present studies provide kinetic conditions for assaying each activity separately (in vivo and in vitro), when both are present in the same membrane preparation. The IIGlc system is responsible for the uptake and phosphorylation of glucose and methyl alpha-glucoside, whereas the IIMan system is less specific and utilizes glucose, mannose, and 2-deoxyglucose. With high sugar concentrations in vitro, IIMan is also capable of phosphorylating methyl alpha-glucoside, fructose, and N-acetylmannosamine, while IIGlc phosphorylates fructose and mannose. The in vivo transport results were qualitatively consistent with the in vitro phosphorylation results, and several of the kinetic parameters also showed good quantitative agreement. The levels of the two activities depended on the growth conditions. In addition, transport studies showed that initial uptake rates of methyl alpha-glucoside and steady state levels of this analogue depended on the energy state of the cells and that these two parameters did not necessarily change in the same direction when metabolic inhibitors were used. A series of E. coli and S. typhimurium mutants were characterized both with respect to their ability to transport the glucose analogues and to phosphorylate them in vitro. The original mutants of Curtis and Epstein, ptsG and ptsM, were found to be defective in II-BGlc and the IIMan complex, respectively.

Biological Transport↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Phosphoryl transfer reactions catalyzed by enzyme I of Salmonella typhimurium.

The phosphorylation of Enzyme I is the first step in the phosphotransfer reaction sequence catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) from Salmonella typhimurium. The characterization of phospho approximately Enzyme I and the reactions in which it participates are described in this report. About 1 mol of phosphoryl group was incorporated per mol of Enzyme I monomer when the homogeneous enzyme was incubated with [32]phosphoenolpyruvate and Mg2+. The phosphoryl group in phospho approximately Enzyme I is linked at the N-3 position in the imidazole ring of a histidine residue. Phospho approximately Enzyme I donates its phosphoryl group to pyruvate (to form phosphoenolpyruvate (P-enolpyruvate)) and to the histidine-containing phosphocarrier protein of the phosphotransferase system (HPr) (to form phospho approximately HPr). In the presence of HPr and appropriate sugar-specific proteins, the phosphoryl group can be transferred from Enzyme I to methyl alpha-glucoside (to form sugar-phosphate). The phosphorylation of Enzyme I by phosphoenolpyruvate requires divalent cation, but the phosphoryl group is transferred from phospho approximately Enzyme I to HPr in the presence of 20 mM EDTA. Kinetic studies show a biphasic rate for Enzyme I phosphorylation, suggesting that the enzyme is phosphorylated in the associated state. Equilibrium experiments were conducted on the following Reactions A and C. (formula: see text). The apparent K' for Reaction B was calculated from K'A and K'C. K'C was found to be about 11. K'A was studied both at very low and high substrate (P-enolpyruvate and pyruvate) concentrations relative to their respective Km values. At low substrate concentrations, the reaction appeared independent of pH in the range of 6.5 to 8.0, and when analyzed according to the simplest expression that could be written for total species of each component (Reaction A), the apparent average K' was 1.5. At high substrate concentrations, about 50% of the Enzyme I was phosphorylated, and this value changed only slightly with large changes in the P-enolpyruvate to pyruvate ratio. Expressions for K'A are derived which partially explain these results by including enzyme-substrate complexes in the equilibrium expression. The K' values were used to derive apparent standard free energy changes for the hydrolysis of the phosphoproteins of the PTS. Since these are similar to those for the hydrolysis of P-enolpyruvate, the phosphate transfer potentials of the PTS phosphoproteins are among the highest of known biological phosphate derivatives. In addition, unlike the reactions which occur during anaerobic glycolysis and electron transport, the high phosphate transfer potential is conserved in the PTS reaction sequence until the last step, the translocation of the sugar substrate across the membrane concomitant with its phosphorylation. Potential regulation of the PTS, in particular the effect of the intracellular ratio of P-enolpyruvate to pyruvate, is considered.

Biological Transport↗