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31.
Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system has two sites of phosphorylation per dimer 总被引:3,自引:0,他引:3
E B Waygood 《Biochemistry》1986,25(14):4085-4090
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. 相似文献
32.
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. 相似文献
33.
34.
Bacterial phosphotransferase system: regulation of mannitol enzyme II activity by sulfhydryl oxidation 总被引:1,自引:0,他引:1
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. 相似文献
35.
Characterization of phosphorylated histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system 总被引:4,自引:0,他引:4
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. 相似文献
36.
Sérgio A Batista Gifone A Rocha Andreia MC Rocha Ivan EB Saraiva Mônica MDA Cabral Rodrigo C Oliveira Dulciene MM Queiroz 《BMC microbiology》2011,11(1):61
Background
Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric cancer and peptic ulcer. Bacterial virulence factors such as CagA have been shown to increase the risk of both diseases. Studies have suggested a causal role for CagA EPIYA polymorphisms in gastric carcinogenesis, and it has been shown to be geographically diverse. We studied associations between H. pylori CagA EPIYA patterns and gastric cancer and duodenal ulcer, in an ethnically admixed Western population from Brazil. CagA EPIYA was determined by PCR and confirmed by sequencing. A total of 436 patients were included, being 188 with gastric cancer, 112 with duodenal ulcer and 136 with gastritis. 相似文献37.
S J Brokx S Napper G Wong A Mirza F Georges L T Delbaere E B Waygood 《Biochimie et biologie cellulaire》1999,77(6):507-513
The structure of the N-terminal domain of enzyme I complexed with histidine-containing protein (HPr) has been described by multi-dimensional NMR. Residues in HPr involved in binding were identified by intermolecular nuclear Overhauser effects (Garrett et al. 1999). Most of these residues have been mutated, and the effect of these changes on binding has been assessed by enzyme I kinetic measurement. Changes to Thr16, Arg17, Lys24, Lys27, Ser46, Leu47, Lys49, Gln51, and Thr56 result in increases to the HPr Km of enzyme I, which would be compatible with changes in binding. Except for mutations to His15 and Arg17, very little or no change in Vmax was found. Alanine replacements for Gln21, Thr52, and Leu55 have no effect. The mutation Lys40Ala also affects HPr Km of enzyme I; residue 40 is contiguous with the enzyme I binding site in HPr and was not identified by NMR. The mutations leading to a reduction in the size of the side chain (Thr16Ala, Arg17Gly, Lys24Ala, Lys27Ala, and Lys49Gly) caused relatively large increases in Km (>5-fold) indicating these residues have more significant roles in binding to enzyme I. Acidic replacement at Ser46 caused very large increases (>100-fold), while Gln51Glu gave a 3-fold increase in Km. While these results essentially concur with the identification of residues by the NMR experiments, the apparent importance of individual residues as determined by mutation and kinetic measurement does not necessarily correspond with the number of contacts derived from observed intermolecular nuclear Overhauser effects. 相似文献
38.
L T Delbaere M Vandonselaar J W Quail E B Waygood J S Lee 《The Journal of biological chemistry》1989,264(31):18645-18646
Single crystals of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli have been grown. This represents one of the first Fab-protein antigen complexes in which the same Fab fragment has previously been crystallized in the uncomplexed state and the structure solved (Prasad, L., Vandonselaar, M., Lee, J. S., and Delbaere, L. T. J. (1988) J. Biol. Chem. 263, 2571-2574). Single crystals up to 0.25 x 0.50 x 0.05 mm in size were grown by the technique of washing and reseeding. The space group is C2, with unit cell dimensions a = 130.0, b = 68.1, and c = 77.6 A; beta = 97.3 degrees; and Z = 4. There is one Fab-histidine-containing protein complex/asymmetric unit, and the solvent content is estimated to be 57%. 相似文献
39.
Some cell cycle events were compared in Chlamydomonas segnis Ettl during its development in synchronous cultures (12:12 LD) supplied with air and air enriched with 5% CO2. In cultures bubbled with air, growth resulted in production of 2 relatively small zoospores. In cultures provided with 5% CO2, 4 large zoospores were formed but not released in darkness unless the cultures were bubbled with CO2-free air and/or exposed to light. Respiration in zoospores was inhibited by high CO2 tension. In cultures maintained under continuous illumination for one cell cycle, provision of 5% CO2 led to enhanced growth, a relatively long S-phase and a 4 h delay of the second cell division. In such cultures, the DNA content of parental cells (12 h L) was insufficient to support two cell divisions. The RNA/DNA ratio of the resulting zoospores was 10 compared to 4 in air cultures. These results provided evidence that the delay of the second cell division was a consequence of the delay in DNA production. 相似文献
40.
Jo EB Halliday Margo E Chase-Topping Michael C Pearce Iain J McKendrick Lesley Allison Dave Fenlon Chris Low Dominic J Mellor George J Gunn Mark EJ Woolhouse 《BMC microbiology》2006,6(1):99-5