19342-33-7Relevant articles and documents
ELECTROCHEMICAL METHODS AND COMPOUNDS FOR THE DETECTION OF ENZYMES
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Page/Page column 19, (2016/04/26)
Disclosed are compositions and methods for the electrochemical detection of enzymes, such as enzymes that are indicative of disease, disorders, or pathogens, such as viruses, bacteria, and fungi, or other disorders. These methods can be used in point-of- care diagnostic assays for the detection of disease, disorder, or pathogen (e.g., to identify the strain of pathogen infecting a patient in a healthcare setting). The electrochemical methods described herein can also be used to assess the susceptibility of a pathogen to an antipathogen drug. Also provided are probes suitable for use in conjunction with the methods described herein.
Enzymatic synthesis of colorimetric substrates to determine α-2,3- and α-2,6-specific neuraminidase activity
Reyes Martinez, Juana Elizabeth,Sardzik, Robert,Voglmeir, Josef,Flitsch, Sabine L.
, p. 21335 - 21338 (2013/11/06)
Glycoconjugates containing either terminal α-2,3- or α-2,6-Neu5Ac-Gal disaccharides are found on cell surfaces of many animal glycans. Each linkage can be specifically recognized by lectins and enzymes such as neuraminidases. Here we describe a one-step enzymatic synthesis of two colorimetric substrates that allow for fast distinction of specific neuraminidase activity. The Royal Society of Chemistry 2013.
Insight into substrate recognition and catalysis by the human neuraminidase 3 (NEU3) through molecular modeling and site-directed mutagenesis
Albohy, Amgad,Li, Matthew D.,Zheng, Ruixiang Blake,Zou, Chunxia,Cairo, Christopher W.
scheme or table, p. 1127 - 1138 (2011/02/23)
The mammalian neuraminidase (NEU) enzymes are found in diverse cellular compartments. Members of the family, such as NEU2 and NEU1, are cytosolic or lysosomal, while NEU3 and NEU4 are membrane-associated. NEU enzymes that act on substrates in the plasma membrane could modulate cellular signaling, cell surface glycoforms and the composition of plasma membrane glycolipids. Therefore, their substrates and mechanism of action are of interest for discerning their physiological roles. We have studied the structure of the human NEU3 using molecular modeling to predict residues involved in the recognition and hydrolysis of glycolipid substrates. To test the model, we have used site-directed mutagenesis of the recombinant protein. Enzymatic studies of the relative activity of these mutants, as well as their pH profiles and inhibition by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, are reported. Using nuclear magnetic resonance spectroscopy, we confirmed that the enzyme is a retaining exo-sialidase, and we propose that the key catalytic residues of the enzyme consist of the general acid-base D50 and the nucleophilic Y370-E225 pair. Mutations of residues expected to interact directly with the sialic acid N5-acetyl (A160, M87, I105) and C7-C9 glycerol side-chain (E113, Y179, Y181) reduced enzymatic activity. We identified several active mutants of the enzyme which contain modifications at the periphery of the active site. Truncations at the N-or C-terminus of more than 10 residues abolished enzyme activity. We propose a catalytic mechanism consistent with the data and identify residues that contribute to glycolipid recognition.
Donor substrate binding to trans-sialidase of Trypanosoma cruzi as studied by STD NMR
Blume, Astrid,Neubacher, Bjoern,Thiem, Joachim,Peters, Thomas
, p. 1904 - 1909 (2008/03/11)
Using STD NMR experiments, we have studied the binding epitopes of p-nitrophenyl glycosides of sialic acid and analogs thereof when bound to Trypanosoma cruzi trans-sialidase (TSia). Time-dependent NMR spectra yielded data on the rate of substrate hydroly
Natural sialoside analogues for the determination of enzymatic rate constants
Indurugalla, Deepani,Watson, Jacqueline N.,Bennet, Andrew J.
, p. 4453 - 4459 (2008/09/19)
Two isomeric 4-methylumbelliferyl-α-d-N- acetylneuraminylgalactopyranosides (1 and 2) were synthesised. These compounds contain either the natural α-2,3 or α-2,6 sialyl-galactosyl linkages, as well as an attached 4-methylumbelliferone for convenient detection of their hydrolyses. These compounds were designed as natural sialoside analogues to be used in a continuous assay of sialidase activity, where the sialidase-catalysed reaction is coupled with an exo-β-galactosidase- catalysed hydrolysis of the released galactoside to give free 4-methylumbelliferone. The kinetic parameters for 1 and 2 were measured using the wild-type and nucleophilic mutant Y370G recombinant sialidase from Micromonospora viridifaciens. Kinetic parameters for these analogues measured using the new continuous assay were in good agreement with the parameters for the natural substrate, 3′-sialyl lactose. Given the selection of commercially available exo-β-galactosidases that possess a variety of pH optima, this new method was used to characterise the full pH profile of the wild-type sialidase with the natural sialoside analogue 1. Thus, use of these new substrates 1 and 2 in a continuous assay mode, which can be detected by UV/Vis or fluorescence spectroscopy, makes characterisation of sialidase activity with natural sialoside linkages much more facile. The Royal Society of Chemistry.
Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase
Kiefel, Milton J.,Wilson, Jennifer C.,Bennett, Simon,Gredley, Matt,Von Itzstein, Mark
, p. 657 - 664 (2007/10/03)
Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase. Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. 1H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme. Copyright (C) 2000 Elsevier Science Ltd.
Effect of neutral pyridine leaving groups on the mechanisms of influenza type A viral sialidase-catalyzed and spontaneous hydrolysis reactions of α-D-N-acetylneuraminides
Chou, Doug T. H.,Watson, Jacqueline N.,Scholte, Andrew A.,Borgford, Thor J.,Bennet, Andrew J.
, p. 8357 - 8364 (2007/10/03)
A reagent panel, comprised of five pyridinium salts of α-D-N-acetylneuraminic acid, was synthesized and then used to probe enzymatic (α-sialidase) and nonenzymatic mechanisms of neuraminide hydrolysis. Spontaneous hydrolysis of the pyridinium salts proceeded via two independent pathways, where unassisted C-N bond cleavage was the rate-determining step. Cationic species (i.e., anomeric carboxylate protonated) displayed apparent pK(a) values in the range of 0.4-0.7. However, spontaneous hydrolyses of the cationic and zwitterionic species had similar β(1g) values of -1.22 ± 0.16 and -1.22 ± 0.07, respectively. The results, plus the activation parameters calculated from the hydrolysis of pyridinium α-D-N-acetylneuraminide (ΔH(+) = 112 ± 2 kJ mol-1 and ΔS(+) = 28 ± 4 J mol-1 K-1), strongly suggest that the anomeric carboxylate does not assist in the departure of neutral pyridine leaving groups. Enzymatic hydrolysis was studied using an influenza viral α-sialidase (A/Tokyo/3/67) which was recombinantly expressed using a baculovirus/insect cell expression system. Sialidase protein was purified by a combination of density gradient centrifugation and gel filtration chromatography. Kinetic parameters for the enzymatic hydrolysis of the pyridinium salts were measured at 37 °C and at pH values of 6.0 and 9.5. The β(1g) values derived for k(cat)/K(m) and k(cat) were essentially zero, indicating that chemical transformations/events are not rate-determining. Rather, this observation is consistent with a model for α-sialidase-catalyzed hydrolyses (Guo, X.; Laver, W. G.; Vimr, E.; Sinnott, M. L., J. Am. Chem. Soc. 1994, 116, 5572) in which k(cat)/K(m) is determined by a conformational change of the first-formed Michaelis complex and k(cat) is determined by the virtual transition state made up of two separate conformational events.
Investigation of the stability of thiosialosides toward hydrolysis by sialidases using NMR spectroscopy.
Wilson,Kiefel,Angus,von Itzstein
, p. 443 - 446 (2008/02/13)
[formula: see text] 1H NMR spectroscopy has been used to investigate whether the alpha(2-->6)-linked thiosialoside 3 and the alpha(2-->3)-linked thiosialoside 9 are hydrolyzed in the presence of Vibrio cholerae sialidase. Similarly, the hydrolysis of the
The N-acetyl neuraminyl oxecarbenium ion is an intermediate in the presence of anionic nucleophiles
Horenstein, Benjamin A.,Bruner, Michael
, p. 1357 - 1362 (2007/10/03)
Solvolysis of CMP N-acetyl neuraminate (CMP-NeuAc) in 1.8 M acetate buffer at pH 5 containing 0.9 M azide results in the formation of both anomers of 2-deoxy-2-azido N-acetyl neuraminic acid in addition to N-acetyl neuraminic acid as determined by 1H-NMR product analysis. A rate dependence on [azide] was observed with an apparent bimolecular rate constant of (2.1 ± 0.30) x 10-3 M-1 min-1 which could only account for half of the azido- NeuAc formed. Comparison of rate, product ratio, and stereochemical data indicate that concurrent pathways for formation of N3-NeuAc are operative, with 17% of product forming from reaction of azide and the tight ion pair. 12% via the solvent separated ion pair, and 6% from the free NeuAc oxocarbenium ion. From the corrected product ratio data, the lifetime of the oxocarbenium ion was estimated to be ≤ 3 x 10-11 s. Solvolysis of CMP- NeuAc at pL. = 5.0 afforded an observed solvent deuterium isotope effect (SDIE) k(H2O)/k(D2O) = 0.45, consistent with specific acid catalysis of glycosidic bond cleavage. A SDIE of 0.66 for the apparent bimolecular azide trapping pathway was also observed. An apparent isotope effect of ~1.1 for trapping of the N-acetyl neuraminyl oxocarbenium ion by water was determined by product analysis of azide trapping in H2O and D2O. An ab initio transition state for attack of water on an N-acetyl neuraminyl oxocarbenium ion model was located which featured a hydrogen bond between the oxocarbenium ion carboxylate and water; proton transfer was not part of the reaction coordinate. It is proposed that the N-acetyl neuraminate carboxylate group stabilizes an intermediate oxocarbenium ion, but the barrier for capture by water is lowered by a transition state hydrogen bond.
