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:4.1.2.42 (DTA)
1,693 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine. 198 Feb 8

Diphtheria toxin (DT) has been studied as a model for understanding active-site structure and function in the ADP-ribosyltransferases. Earlier evidence suggested that histidine-21 of DT is important for the ADP-ribosylation of eukaryotic elongation factor 2 (EF-2). We have generated substitutions of this residue by cassette mutagenesis of a synthetic gene encoding the catalytic A fragment (DTA) of DT, and have characterized purified mutant forms of this domain. Changing histidine-21 to alanine, aspartic acid, leucine, glutamine, or arginine diminished ADP-ribosylation activity by 70-fold or greater. In contrast, asparagine proved to be a functionally conservative substitution, which reduced ADP-ribosylation activity by < 3-fold. The asparagine mutant was approximately 50-fold-attenuated in NAD glycohydrolase activity, however. Dissociation constants (Kd) for NAD binding, determined by quenching of the intrinsic protein fluorescence, were 15 microM for wild-type DTA, 160 microM for the asparagine mutant, and greater than 500 microM NAD for the alanine, leucine, glutamine, and arginine mutants. These and previous results support a model of the ADP-ribosylation of EF-2 in which histidine-21 serves primarily a hydrogen-bonding function. We propose that the pi-imidazole nitrogen of His-21 hydrogen-bonds to the nicotinamide carboxamide, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism, and then stabilizing the transition-state intermediate of this reaction.
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PMID:Active-site mutations of the diphtheria toxin catalytic domain: role of histidine-21 in nicotinamide adenine dinucleotide binding and ADP-ribosylation of elongation factor 2. 817 90

Two novel complexes of [Cu(HAsp)ImH2O]SO4 x 4 H2O and [Cu(Asp)Im(OH)] x 4 H2O (HAsp = Aspartic acid molecule, Asp = Aspartic acid ion, Im = Imidazole) were synthesized and characterized by elemental analysis, IR spectroscopy and TG-DTA. The interactions of the complexes with sperm DNA were studied by electronic absorption and fluorescence spectra. Results showed that the reactions of the two complexes with DNA are obviously different: for [Cu(HAsp)ImH2O]SO4 x 4 H2O, it is intercalation companied by electrostatic effect, while [Cu(Asp)Im(OH)] x 4 H2O mainly cooperates with the nitrogen atom of the DNA base pair, which induces the breakage of DNA double helix. The reasons for these differences in their DNA binding modes were also discussed.
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PMID:[Synthesis and DNA binding spectroscopic studies of Cu(II)-HAsp-Im complexes]. 1751 61

The action of anthrax toxin relies in part upon the ability of the protective antigen (PA) moiety to form a heptameric pore in the endosomal membrane, providing a portal for entry of the enzymic moieties of the toxin into the cytosol. Pore formation is dependent on a conformational change in the heptameric prepore that occurs in the neutral to mildly acidic pH range, and it has been hypothesized that protonation of one or more histidine residues triggers this transition. To test this hypothesis, we used biosynthetic methods to incorporate the unnatural amino acid analogue 2-fluorohistidine (2-FHis) into PA. 2-FHis is isosteric with histidine but resists protonation at physiological pH values due to a dramatically reduced side-chain pKa ( approximately 1). We found that 2-FHis-labeled PA was biologically inactive, as judged by its inability to deliver a model intracellular effector, LFN-DTA, to the cytosol of CHO-K1 cells. However, whereas 2-FHis blocked a conformational transition in the full-length PA83 protein in the pH 5-6 range, the pH dependence of prepore-to-pore conversion of (PA63)7 was unchanged from the wild-type protein, implying that this conversion is not dependent on His protonation. Consistent with this result, the labeled, trypsin-activated PA was able to permeabilize liposomes to K+ and retained pore-forming activity in planar phospholipid bilayers. The pores in planar bilayers were incapable, however, of translocating a model ligand in response to a transmembrane pH gradient or elevated voltage. The results indicate that protonation of residues other than His, presumably Glu and/or Asp side chains, triggers pore formation in vitro, but His residues are nonetheless important for PA functioning in vivo.
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PMID:Effect of 2-fluorohistidine labeling of the anthrax protective antigen on stability, pore formation, and translocation. 1804 73

Single crystals of pure and L-aspartic acid doped Zinc (Tris) Thiourea Sulphate (ZTS) were grown from aqueous solution by solution growth method. The cell parameters and structure of the grown crystals were determined by X-ray diffraction studies. The presence of functional group in the compound has been confirmed by FTIR and FT-Raman analysis. The optical transparency range has been studied through UV-Vis spectroscopy. TGA/DTA studies show thermal stability of the grown crystals. Microhardness study reveals that the hardness number (Hv) increases with load for pure and doped ZTS crystals. Dielectric studies have been carried out and the results are discussed. The second harmonic generation was confirmed for l-aspartic acid doped ZTS which is greater than pure ZTS.
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PMID:Effect of L-aspartic acid on the growth, structure and spectral studies of Zinc (tris) Thiourea Sulphate (ZTS) single crystals. 2489 30

Antiepileptic drug (Preg) and neurotransmitters (Gpn, Gly, Asp, Glu, Ser, GABA and Ade) were used to synthesis a series of ternary Cu²⁺ complexes. Surface morphology and chemical composition of the complexes were using studied SEM and EDX spectra. Purity, molecular weight and formulae of the complexes were determined from GC-MS spectra and elemental analysis. XRD data and N-Treor implemented in Expo2014 computer program reveal monoclinic crystal system with space group P1 2/c 1 and P 1 21 1 of the complexes. IR spectra exhibited that Preg, Gpn and GABA coordinated to the Cu²⁺ as monodentate ligand through COOH whereas the amino acids bonded through the -NH₂, COO- groups. UV-Vis spectra and magnetic moment values indicated pseudo tetrahedral stereochemistry. ESR spectra showed that the complexes have isotropic and axial structures with d_x²_-y² ground state. TGA, DTG and DTA confirm the suggested structure and mechanism for thermal decomposition was suggested. Kinetic and thermodynamic parameters were calculated using Coats-Redfern equation. The complexes [Cu(Preg)(Ser)Cl] and [Cu(Preg)(Ade)Cl₂] showed greater anticonvulsant activity compared against PTZ-induced seizures in male Albino mice. Recorded latency time for the complexes [Cu(Preg)(Ser)Cl] and [Cu(Preg)(GABA)(OH)Cl].2H₂O was longer than that recorded with Preg indicating higher anticonvulsant effect.
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PMID:Binary and ternary Cu(II) complexes of pregabalin with excitatory and inhibitory neurotransmitters and their antiepileptic effect. 3220 80

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