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E B Waygood

Publications and source records attributed to E B Waygood.

68 records · Page 4Linked to original sources
PubMed1980

Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. Purification to homogeneity and some properties.

Enzyme I of the phosphoenolpyruvate - sugar phosphotransferase system (PTS) has been purified to homogeneity from Escherichia coli. A merodiploid strain P650 which had an extra copy of the gene for enzyme I resulting in a twofold increase in the amount of activity was used. The enzyme is a dimer of 67 000 +/- 5000 molecular weight subunits. At low protein concentration and 4 degrees C the monomer predominates, while at room temperature the dimer predominates. At higher protein concentrations (2 to 10 mg) this reversible temperature-dependent association-dissociation is not found. Enzyme I has a pH optimum of pH 7.2, a Km for HPr of 9 +/- 3 microM, a Km for phosphoenolpyruvate of 0.18 +/- 0.04 mM, and kinetics that are consistent with a bi bi Ping-Pong mechanism. No allosteric regulation of kinetic activity has been found. The amino acid composition has been determined and the epsilon 1% 280 nm is 4.4. Evidence suggests that the phosphorylated form of enzyme I is more stable.

Cell Division↗
PubMed1980

Resolution of the phosphoenolpyruvate: fructose phosphotransferase system of Escherichia coli into two components: enzyme IIfructose and fructose-induced HPr-like protein (FPr).

A protein that substitutes for histidine-containing protein (HPr) in the phosphoenolpyruvate, fructose phosphotransferase system has been found in Escherichia coli grown on fructose. The impure preparation of the fructose-induced HPr-like protein (FPr) appears to be an extrinsic membrane protein which differs from HPr on the basis of its apparent molecular weight (45 000 vs. 9600, respectively), its affinity for DEAE-cellulose and its ability to promote sugar phosphorylation which is specific for fructose, rather than for glucose.

Bacterial Proteins↗
PubMed1976

The control of pyruvate kinase of Escherichia coli. Binding of substrate and allosteric effectors to the enzyme activated by fructose 1,6-bisphosphate.

The binding of various regulatory ligands and substrates to the fructose bisphosphate activated pyruvate kinase from Escherichia coli has been studied at equilibrium. The allosteric activator, fructose bisphosphate, and the substrate phosphoenolypyruvate bind in a cooperative manner to the enzyme. There is one site for each of these ligands per monomer. In the presence of fructose bisphosphate the binding of phosphoenolpyruvate follows an absorption isotherm, i.e., all homotropic interactions of the substrate are lost. In reciprocal experiments, however, both phosphoenolpyruvate and KCl are required in order to facilitate binding of the activator. The allosteric inhibitors of pyruvate kinase, ATP, succinyl-CoA, and GTP compete on the enzyme surface with the binding of the activator, fructose bisphosphate, Inhibitor pairs such as ATP and succinyl-CoA together bring about not cooperative but only additive inhibition of the binding of the activator. The nucleotide substrate GDP and the allosteric inhibitor GTP have in contrast to the activator two seemingly noninteracting sites on each monomer. In the saturating presence of fructose bisphosphate, however, binding of GDP and possibly also of GTP occurs at only one site on each monomer. Magnesium ions inhibit binding of GDP and GTP. KCl which is an activator of the enzyme along with its analogues, such as ammonia, thallium, rubidium, etc., enhances the binding of phosphoenolpyruvate but not of the nucleotides or fructose bisphosphate. The data are analyzed on the basis of a two-site model, where the substrate and fructose bisphosphate bind to one conformation and the inhibitors to the other.

Allosteric Regulation↗
PubMed1975

The control of pyruvate kinases of Escherichia coli. II. Effectors and regulatory properties of the enzyme activated by ribose 5-phosphate.

The pyruvate kinases of Escherichia coli activated by ribose 5-phosphate (RP) has been partially purified. The active form of the enzyme has a molecular weight of about 180 000 as judged by sucrose density gradient centrifugations and Sephadex G-150 chromatography. On dissociation in the absence of sulfhydryl reagents such as dithiothreitol, the enzyme is inactivated and it has a molecular weight of about 110 000. Various substrates and effectors of the enzyme, with the exception of phosphate, do not influence the association-dissociation equilibrium of the enzyme. The enzyme, unlike pyruvate kinases from many other sources, is not activated by potassium ions. Sulfate and phosphate ions are inhibitory to the enzyme. Phosphate seems to be an allosteric inhibitor and its effect is completely antagonized by activators. The enzyme is activated in an allosteric manner by two classes of compounds, nucleoside monophosphates and sugar phosphates of the hexose monophosphate pathway. Amongst the nucleotides, guanosine 5'-phosphate and adenosine 5'-phosphate are the most effective activators. Amongst the hexose monophosphate pathway intermediates, RP is the most powerful activator, with apparent activation constants as low as 1 Mu. Sugar phosphates esterified at C-1 or both terminal positions are entirely ineffective in activation. The effectors act by changing the Michaelis constant for the substrates. Both of the substrates of the enzyme, adenosine diphosphate and phosphoenolpyruvate, yield cooperative-concentration plots in the presence of unsaturating concentrations of the fixed changing substrate. The initial velocity plots for both substrates become hyperbolic in the presence of saturating concentrations of RP.

Adenosine Diphosphate↗
PubMedDate not supplied

A novel phosphoprotein dependent on the bacterial phosphoenolpyruvate-sugar phosphotransferase system.

A protein has been fond by isoelectricfocusing and autoradiography in Escherichia coli and Salmonella typhimurium which was phosphorylated by enzyme I and an histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate-sugar phosphotransferase system (PTS). This protein was not factor III glc nor was it specifically induced by fructose. Its presence in soluble crude extracts was dependent upon growth conditions; however, the two bacteria had different patterns and amounts in respect to this novel protein. The protein was present in S. typhimurium SB2950 which has an extensive deletion through the pts operon, thus indicating that it must be coded for elsewhere on the genome.

Bacterial Proteins↗
PubMedDate not supplied

Properties of phosphorylated protein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

The phosphohydrolysis properties of the following phosphoprotein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) were investigated: enzyme I, HPr, and the IIAGlc domain of the glucose enzyme II of Bacillus subtilis; and IIAGlc (fast and slow forms) of Escherichia coli. The phosphohydrolysis properties were also studied for the site-directed mutant H68A of B. subtilis IIA Glc. Several conclusions were reached. (i) The phosphohydrolysis properties of the homologous phosphoprotein intermediates of B. subtilis and E. coli are similar. (ii) These properties deviate from those of isolated N delta 1- and N epsilon 2-phosphohistidine indicating the participation of neighbouring residues at the active sites of these proteins. (iii) The rates of phosphohydrolysis of the H68A mutant of B. subtilis IIAGlc were reduced compared with the wild-type protein, suggesting that both His-83 and His-68 are present at the active site of wild-type IIAGlc. (iv) The removal of seven N-terminal residues of E. coli IIAGlc reduced the rates of phosphohydrolysis between pH 5 and 8.

Bacillus subtilis↗
PubMedDate not supplied

Structural comparison of the histidine-containing phosphocarrier protein HPr.

The phosphocarrier protein HPr is a central component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) that is responsible for carbohydrate uptake in many bacterial species. A number of three-dimensional structures of HPrs from both Gram-positive and Gram-negative bacteria have been determined; the overall folding topology of HPr is an open-faced beta-sandwich composed of three alpha-helices and a beta-sheet. A detailed structural comparison of these HPrs has been carried out. Besides the overall main chain folding, many detailed structural features are well conserved in all HPr structures. The three x-ray structures of HPrs from Escherichia coli, Streptococcus faecalis, and Bacillus subtilis show considerable overall similarity with respect to the positions of the C alpha atoms. A significant structural difference between HPrs from Gram-positive and Gram-negative bacteria is found in the region of Gly54, owing to the steric effects of Tyr37 in HPrs from the Gram-positive species. The region around Gly54 is involved in the binding of HPr to other PTS proteins and the differences in this region may be responsible for some of the poor functional complementation between HPrs from Gram-positive and Gram-negative species. The active center region, residues 12-18, appears to have significant differences in the comparisons between the overall structures. These differences support the proposal that phosphorylation and dephosphorylation of the active site His15 is accompanied by conformational changes. However, a local structural comparison of residues 12-18 from the x-ray structures of HPrs from E. coli and B. subtilis, and the two-dimensional nuclear magnetic resonance structure of B. subtilis HPr suggests that there is a conserved active center involving residues His15, Arg 17, and Pro18, which shows little conformational change during the phosphorylation cycle. The results of other experimental approaches, including site-directed mutagenesis and NMR spectroscopy, are in some cases difficult to rationalize with some of the details of the structures, but do appear to favour the conclusion that little conformational change occurs.

Amino Acid Sequence↗