Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tyrosine 121, a residue located in a alpha-helical polypeptide segment of glucosamine 6-phosphate deaminase from Escherichia coli, has recently been proposed to have a role in the binding of the allosteric activator N-acetyl-D-glucosamine 6-phosphate. Accordingly, the site-directed mutants Tyr 121-Thr and Tyr 121-Trp were constructed, to assess experimentally the role of Tyr 121 in the allosteric function of the enzyme. The kinetic study of both mutant forms revealed that the replacements caused striking changes in allosteric activator binding and allosteric properties, when compared to the wild-type enzyme. While the wild-type deaminase behaves as a classical allosteric K-system which can be described by the allosteric concerted model, both mutant forms present an asymmetric behavior toward the allosteric activator, which can be described as two distinct half-of-the-sites allosteric activation steps occurring with different affinities for the N-acetyl-D-glucosamine 6-phosphate. During the first (high affinity) activation phase, the mutant forms of deaminase behave as mixed K/V allosteric enzyme. The biphasic activation curve was also demonstrated by direct binding measurements of the 14C-labeled activator to Tyr 121-Trp and Tyr 121-Thr deaminases. The kinetic analysis of these mutant forms also showed that the threonine replacement produced an important distortion of the enzyme structure reflected in a considerable decrease of its catalytic efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Asymmetric allosteric activation of Escherichia coli glucosamine-6-phosphate deaminase produced by replacements of Tyr 121. 774 11

N-Acetyl-D-[2-3H]glucosamine was synthesized from N-acetyl-D-mannosamine by alkaline 2-epimerization in pyridine containing 3H2O and nickelous acetate. The reaction involves reversible formation of an enol intermediate and therefore also resulted in incorporation of tritium into N-acetylmannosamine. After completed reaction, the two N-acetylhexosamines were separated from other radioactive products and Morgan-Elson chromogens by chromatography on a column of Sephadex G-10, which was eluted with 10% ethanol, and were then separated from each other by chromatography on Sephadex G-15 in 0.27 M sodium borate (pH 7.8). The location of the incorporated tritium was established by treatment of the N-acetylhexosamines with borate under the conditions of the Morgan-Elson reaction, which converts the sugars to Kuhn's chromogen I with concomitant loss of the C-2 hydrogen. As expected, this treatment resulted in the formation of 3H2O, indicating that the tritium was located at C-2. [2-3H]Glucosamine was prepared by acid hydrolysis of the labelled N-acetylglucosamine and was converted to [2-3H]glucosamine 6-phosphate by incubation with hexokinase and ATP. The sugar phosphate was used as a substrate for glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10) in a simple 3H2O release assay.
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PMID:Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water. 778 Jan 91

The glycosylation pattern of the external envelope glycoprotein of human immunodeficiency virus type 2 (HIV-2) was studied in dependence on host cells and virus isolates. Strains HIV-2ALT, HIV-2ROD and HIV-2D194, differing in their biological properties and in the amino acid sequences of their env genes, were propagated in MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytes and monocytes/macrophages in the presence of [6-3H]glucosamine. Radiolabelled viral glycoproteins were isolated from the cell-free supernatants and digested with trypsin. Glycans were sequentially liberated by endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F, and fractionated according to charge and size. Comparison of the oligosaccharide profiles revealed that the envelope glycoproteins of different virus isolates, propagated in the same host cells, yielded very similar glycan patterns, whereas cultivation of an isolate in different host cells resulted in markedly divergent oligosaccharide maps. Variations concerned the proportion of high-mannose-, hybrid- and complex-type substituents, as well as the state of charge and structural parameters of the complex-type species. As a characteristic feature, complex-type glycans of macrophage-derived viral glycoprotein were almost exclusively substituted by lactosamine repeats. Hence, glycosylation of the HIV-2 external envelope glycoprotein seems to be primarily governed by host cell-specific factors rather than by the amino acid sequence of the corresponding polypeptide backbone.
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PMID:Oligosaccharide profiles of HIV-2 external envelope glycoprotein: dependence on host cells and virus isolates. 782 9

The interaction of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli with its allosteric activator, N-acetyl-D-glucosamine 6-phosphate, was studied by different spectrophotometric methods. Analysis of the circular-dichroism differential spectra produced by the binding of the allosteric activator or the competitive inhibitor 2-amino-2-deoxy-D-glucitol 6-phosphate (a homotropic ligand displacing the allosteric equilibrium to the R conformer), strongly suggests the presence of tyrosine residues at or near the allosteric site, although a conformational effect cannot be ruled out. The involvement of a single tyrosine residue in the N-acetyl-D-glucosamine-6-phosphate binding site of glucosamine-6-phosphate deaminase was supported by spectrophotometric pH titrations performed in the presence or absence of the homotropic and heterotropic ligand. In these experiments, a single titrated tyrosine residue is completely protected by saturation with the allosteric activator; this group is considerably acidic (pK 8.75). The analysis of the amino acid sequence of the deaminase using a set of indices for the prediction of surface accessibility of amino acid residues, suggests that the involved residue may be Tyr121 or Tyr254.
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PMID:Spectrochemical evidence for the presence of a tyrosine residue in the allosteric site of glucosamine-6-phosphate deaminase from Escherichia coli. 812 98

The pathways of glucose utilization for energy production in the malaria parasite, Plasmodium falciparum, have been studied extensively. Little is known, however, about the reactions by which glucose is converted into complex carbohydrates in the parasite, and knowledge of the catabolism of these substances is likewise scanty. The present investigation was undertaken to determine whether the parasites possess a key enzyme of glucosamine catabolism, i.e. glucosamine 6-phosphate deaminase (EC 5.3.1.40), which catalyses the conversion of the sugar phosphate to fructose 6-phosphate and ammonia. Lysates of Plasmodium-infected erythrocytes had substantially higher deaminase activity than control samples from normal erythrocytes, and an even higher specific activity was observed in extracts of isolated parasites, amounting to 20-40 times that of uninfected cells. Anion exchange chromatography indicated that the parasite deaminase eluted in a retarded position when compared to the elution profile of the erythrocyte enzyme. The charge difference suggested by these findings was established more directly by chromatofocusing, which indicated pI values of 6.85 and 8.55 for the parasite and erythrocyte deaminases, respectively. Other differences were also observed, notably a greater thermolability on the part of the parasite enzyme. These results indicated that the parasites synthesize a specific deaminase that is distinct from the normal erythrocyte enzyme. Studies on synchronized parasite cultures further indicated that the parasite deaminase is developmentally regulated, because a dramatic increase in activity levels occurred during the later stages of parasite development.
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PMID:Glucosamine 6-phosphate deaminase in Plasmodium falciparum. 855 58

The cell wall structure of Salmonella typhimurium has been studied for the first time during transit from free-living to parasitic lifestyles. Peptidoglycan of S. typhimurium proliferating within human epithelial cells contains a high proportion of previously unidentified muropeptides (5-10-fold higher than in extracellular bacteria). Amino acid and mass-spectrometry analyses showed that these new components consist of dimeric cross-linked muropeptides lacking one of the two disaccharide (N-acetyl-glucosamine-beta-(1-->4)-N-acetyl-muramic acid) molecules. This unique structure suggests an active role for an N-acetyl-muramyl-L-alanine-amidase in remodelling the peptidoglycan of intracellular S. typhimurium. Additional alterations observed included: (i) the absence of glycine-containing muropeptides; (ii) the increase in the relative proportion of muropeptides cross-linked by L(meso)-diaminopimelyl-D(meso)-diaminopimelic acid (L-D) peptide bridges; and, (iii) the decrease in the global cross-linkage of the macromolecule. The structural alterations observed in the peptidoglycan of intracellular bacteria do not produce loss of the cell envelope. These results show that intracellular residence of S. typhimurium within epithelial cells is accompanied by significant changes in the bacterial cell wall. Remodelling of peptidoglycan structure may constitute another sophisticated strategy of this pathogen for adapting to and colonizing the intracellular niche of eukaryotic cells.
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PMID:Peptidoglycan structure of Salmonella typhimurium growing within cultured mammalian cells. 915 41

Glucosamine-6-phosphate deaminase (GNPDA) catalyzes the conversion of glucosamine-6-phosphate to fructose-6-phosphate, a reaction that under physiological conditions proceeds to the formation of fructose-6-phosphate. Though first identified in mammalian tissues in 1956, the enzyme has not previously been molecularly characterized in mammalian tissues, although a bacterial GNPDA has been cloned. Recently, a protein displaying similarity to bacterial GNPDA was purified and cloned from sperm extract. It was proposed that this protein was the factor, found in sperm extracts, that causes calcium oscillations in cells; thus, the protein was named 'oscillin.' We demonstrate that oscillin is the mammalian form of glucosamine 6-phosphate deaminase by showing that cloned oscillin has a robust GNPDA activity and can account for all such activity in mammalian tissues extracts. In situ hybridization and immunohistochemistry localize GNPDA selectively to tissues with high energy requirements such as the apical zone of transporting epithelia in the proximal convoluted tubules of the kidney and the small intestine; to neurons (but not glia) and especially to nerve terminals in the brain; and to motile sperm. Recombinant GNPDA and GNPDA purified to homogeneity from hamster sperm fail to elevate intracellular calcium when injected into mouse eggs over a wide range of concentrations under conditions in which sperm extracts elicit pronounced calcium oscillations. Thus, the calcium-releasing or oscillin activity of sperm extracts is due to a substance other than GNPDA. Since GNPDA is the sole enzyme linking hexosamine systems with glycolytic pathways, we propose that it provides a source of energy in the form of phosphosugar derived from the catabolism of hexosamines found in glycoproteins, glycolipids, and sialic acid-containing macromolecules. Evidence that GNPDA can regulate hexosamine stores comes from our observation that transfection of GNPDA into HEK-293 cells reduces cellular levels of sialic acid.
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PMID:Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity. 943 14

The involvement of tyrosine residues in the allosteric function of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli was first proposed on the basis of a theoretical analysis of the sequence and demonstrated by spectrophotometric experiments. Two tyrosine residues, Tyr121 and Tyr254, were indicated as involved in the mechanism of cooperativity and in the allosteric regulation of the enzyme [Altamirano et al. (1994) Eur. J. Biochem. 220, 409-413]. Tyr121 replacement by threonine or tryptophan altered the symmetric character of the T --> R transition [Altamirano et al. (1995) Biochemistry 34, 6074-6082]. From crystallographic data of the R allosteric conformer, Tyr254 has been shown to be part of the allosteric pocket [Oliva et al. (1995) Structure 3, 1323-1332]. Although it is not directly involved in binding the allosteric activator, N-acetylglucosamine 6-phosphate, Tyr 254 is hydrogen bonded through its phenolic hydroxyl to the backbone carbonyl from residue 161 in the neighboring polypeptide chain. Kinetic and binding experiments with the mutant form Tyr254-Phe of the enzyme reveal that this replacement caused an uncoupling of the homotropic and heterotropic effects. Homotropic cooperativity diminished and the allosteric activation pattern changed from one of the K-type in the wild-type deaminase to a mixed K-V pattern. On the other hand, Tyr254-Trp deaminase is kinetically closer to a K-type enzyme and it has a higher catalytic efficiency than the wild-type protein. These results show that the interactions of Tyr254 are fundamental in coupling binding in the active site to events occurring in the allosteric pocket of E. coli glucosamine 6-P deaminase.
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PMID:Tyr254 hydroxyl group acts as a two-way switch mechanism in the coupling of heterotropic and homotropic effects in Escherichia coli glucosamine-6-phosphate deaminase. 960 Oct 45

Transfer of truncated oligosaccharides to yeast exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated. When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme. As expected, none of the latter had affinity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc. Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N4-N-acetyl-beta-glucosamine asparagine amidase. These results unambiguously demonstrate that GlcNAc2 can be transferred from dolichol-PP-GlcNAc2 to one or both sequons of yeast Exg. Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum.
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PMID:N-glycosylation by transfer of GlcNAc2 from dolichol-PP-GlcNAc2 to the protein moiety of the major yeast exoglucanase. 967 21

The enzyme glucosamine-6-phosphate deaminase from beef kidney has been purified to homogeneity by allosteric-site affinity chromatography. Its amino acid composition and the N-terminal sequence (1-42), were obtained. The amino acid sequence of this segment is essentially identical to the corresponding regions of the human and hamster glucosamine-6-phosphate deaminases. The beef enzyme is a hexamer of 32.5 kDa subunits; this is nearly 2.5 kDa higher than the molecular mass of the homologous enzyme from Escherichia coli. Beef kidney deaminase exhibits a notable difference from the bacterial enzyme in its allosteric activation by N-acetylglucosamine 6-phosphate This metabolite, which is also is the allosteric activator of the bacterial glucosamine-6-phosphate deaminase, activates the enzyme by increasing its kcat without any change in the Km values for glucosamine 6-phosphate, over a wide range of activator concentration. This observation places beef kidney deaminase in the class of V-type allosteric systems.
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PMID:Glucosamine-6-phosphate deaminase from beef kidney is an allosteric system of the V-type. 977 1


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