No description available.

No description available.

No description available.

No description available.

No description available.

The Compounds 2-deoxy-2-fluoro-β-D-glucosyl fluoride (1), 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2), 2-deoxy-2-fluoro-β-D-galactosyl fluoride (3) and 2-deoxy-2-fluoro-β-D-mannosyl fluoride (4) were all found to be potent covalent inhibitors of β-glucosidase from Alcaligenes faecalis (pABG5 β-glucosidase), which function through the accumulation of a stable glycosyl-enzyme intermediate. The mechanism of action for these inhibitors was investigated and found to be both specific and active site directed, involving a 1:1 stoichiometric formation of an enzyme inhibitor complex. Investigation of the pre-steady state kinetics for the inhibition reaction provided values for the rates of formation (kon) and hydrolysis (koff) of the glycosylated-enzyme intermediate. The values of kon determined in this manner are 5.9, 25, 3.6 and 5.6 min.⁻¹ for compounds 1,2,3 and 4 respectively, while the values of koff were found to be much smaller; 1.5x10⁻⁵, 5.4 x 10⁻³ and 1.0 x 10⁻³ min.⁻¹, respectively for the intermediates formed from compounds 1, 3 and 4. The isolated intermediates were also found to be competent in the catalysis of transglycosylation reactions and evidence for the participation of a specific binding site for the acceptor compound in this process was gained. The results of these kinetic experiments were corroborated by data obtained from ¹⁹F-NMR spectra of the glycosyl-enzyme intermediate and by isolation and subsequent characterization of the transglycosylation products. The stereochemistry of the inhibition reaction was investigated by a simple ¹⁹F-NMR experiment, and was found to be consistent with a double displacement reaction mechanism, as would be expected from the generally accepted reaction mechanism for this type of enzyme. The compound D-glucal was found to be a substrate for pABG5 β-glucosidase (kcat = 2.28 min.⁻¹ Km = 0.85 mM). Hydration of this compound by pABG5 β-glucosidase in deuterated buffers demonstrated that the double bond of D-glucal was deuterated stereo-specifically from below the α-face. The compound 2-fluoro-D-glucal was found to be a weak competitive inhibitor (Ki = 30 mM) of pABG5 β-glucosidase. Glycogen phosphorylase catalyses the reversible phosphorolysis of glycogen. A series of deoxy analogues of the enzymes natural substrate, α-D-glucose 1-phosphate have been prepared and along with a number of deoxyfluoro analogues tested as substrates. All were found to act as substrates but at exceedingly slow rates. The large rate reductions when compared with the normal substrate can be attributed to a deleterious combination of electronic and binding effects in the modified substrates reducing the stability of the enzymic transition states. A linear free energy relationship between kcat and the first order rate constant for the acid catalysed hydrolysis of the same series of deoxy and deoxyfluoro glucopyranosyl phosphates was demonstrated, suggesting similar transition states for the two reactions and implicating an oxocarbonium ion-like transition state in the enzymic reaction. The binding data obtained from the steady state kinetics of these analogues suggests that hydrogen bonding interactions are qualitatively conserved in the glucopyranose binding site during the T- to R-state conformational transition of the enzyme and that interactions between the enzyme and the hydroxyl groups at the 3- and 6-positions of the glucopyranose ring of the substrate are potentially important for stabilization of the enzymic transition state. The specificity of the substrate phosphate binding site has been probed using the compounds 2-deoxy-2-fluoro-α-D-glucopyranosyl phosphate (5), (1-deoxy-α-D-glucopyranosyl) methylphosphonate (6) and 2-deoxy-2-fluoro-α-D-glucopyranosyl phosphofluoridate (7). The results suggested that phosphorylase b can bind both the monanionic and dianionic forms of its substrate with approximately equal affinity. NMR studies of the ternary enzyme-ligand complexes formed with glycogen phosphorylase b and 5 or 6 indicated that no proton donation occurred in the ground-state active site complex. A preliminary investigation into the ability of the cellulase complex from a number of different wood-degrading fungi to hydrolyse p-nitrophenyl β-glucoside has been carried out. This work is aimed at producing environmentally safe fungicides, which are activated by the β-glucosidase component of the cellulase complex in these organisms and this study was carried out in conjunction with Forintek Canada. / Science, Faculty of / Chemistry, Department of / Graduate

No description available.

No description available.

N-linked glycosylation is the most common form of post-translational modification in nature and is essential to almost all enveloped viruses, including members of the Flaviviridae family. The host cell N-linked glycoprotein processing pathway is utilised by these viruses and as such has long been identified as a potential target for the development of antiviral drugs. Here, the antiviral mechanisms of three classes of small molecules targeting the secretory pathway and altering viral envelope glycosylation are investigated, using the HCV surrogate model, BVDV. The antiviral activity of imino sugars, principally through α-glucosidase inhibition, is well-characterised and here, a group of novel adamantyl coupled imino sugars are investigated and demonstrated to inhibit ER α glucosidases, which correlates with their antiviral activity against BVDV. Additionally, BVDV is used to study the antiviral mechanism of action of nitazoxanide. Nitazoxanide, the parent compound of the thiazolide class of structures, is a broadly antimicrobial compound with antiviral activity against HBV, HCV, influenza, JEV and others. Here, nitazoxanide is shown to be antiviral against BVDV by inducing Ca²⁺ release from ATP-sensitive intracellular calcium stores, disrupting ER-Golgi trafficking and inhibiting complex glycan formation. Finally, the potential of Golgi endo-α-mannosidase as an antiviral target is explored, using the endomannosidase inhibitor glucose-isofagomine in conjunction with the imino sugar α-glucosidase inhibitor NAP-DNJ. Endomannosidase is shown to be a valid antiviral target for BVDV, both alone and in combination with α-glucosidase inhibition, and is utilised by viral glycoproteins to acquire complex glycan structure, even in the absence of α-glucosidase inhibition. Altogether, this work furthers our understanding of the varied antiviral mechanisms of small molecules targeting the secretory pathway, enhancing the search for novel antiviral drugs directed against host cell machinery.

Glicoconjugados são abundantes na superfície de Trypanosoma cruzi e têm sido bastante estudados por diferentes grupos. A degradação dessas moléculas, no entanto, tem sido alvo de pouco interesse. O objetivo deste trabalho foi determinar a atividade de alfa-L-fucosidase em T.cruzi uma vez que trabalhos anteriores haviam concluído que várias hidrolases, entre elas a alfa-L-fucosidase estavam ausentes em epimastigota (AVILA, et al., 1979). Empregando-se p-nitrofenilfucopiranosídeo como substrato e extrato de formas epimastigotas, verificou-se que a enzima apresenta praticamente a mesma atividade em um intervalo de pH entre 6,0 e 7,5, caindo drasticamente em pHs mais ácidos. A incubação prévia da enzima a 28°C em pH 7,0 leva à perda de aproximadamente 30% de sua atividade após 1h 30mim e à perda de 100% após 4 horas de incubação. O efeito de íons na atividade da enzima foi estudado,verificando-se que Zn +2 inibe 90% sua atividade, enquanto que outros, como o Ca +2 praticamente não tem efeito. A enzima é parcialmente encontrada na fração particulada, podendo ser solubilizada parcialmente com 1% de Triton X-100 ou com NaCl 1 M. As tentativas feitas de purificar a enzima foram infrutíferas, uma vez que não se encontraram condições para manter a proteína ativa por longos períodos de tempo. A alfa-L-fucosidase está presente não só em pimastigotas, mas também em tripomastigotas, embora parentemente com diferentes atividades específicas, sendo maior em epimastigotas. Mesmo nos epimastigotas, grandes variações de atividade específica foram detectadas ao longo deste trabalho (de 0,03 a 0,23 unidades). Anticorpos preparados contra alfa-L-fucosidase comercial de epidídimo bovino imunoprecipitaram de extratos de epimastigotas previamente marcados com 35 S-metionina, um polipeptídeo em torno de 50 kDa após eletroforese em gel desnaturante e uma banda de 130-150 kDa em gel não desnaturante, sugerindo que a enzima em T.cruzi pode ser dimérica, a exemplo de outras alfa-L-fucosidases descritas na literatura. A imunoprecipitação de extrato de epimastigotas marcados com 35 S-metionina na presença de tunicamicina, com o anticorpo anti-alfa-L-fucosidase revelou um polipeptídeo de 45 kDa, mostrando que a enzima é glicosilada. A glicosilação daenzima também foi observada pelo emprego de corantes comerciais. Além disso, os anticorpos anti-alfa-L-fucosidase imunoprecipitam moléculas com atividade de alfa-L-fucosidase,embora não se tenha observado aumento da atividade, possivelmente devido à perda de atividade da enzima nas condições empregadas durante a imunoprecipitação. Os anticorpos anti-alfa-L-fucosidase reconhecem, por imunofluorescência indireta, tanto as formas epimastigotas como tripomastigotas de cultura de tecido. A análise por microscopia de transmissão mostra a reatividade intensa do anticorpo com uma região membranar localizada na região posterior do epimastigota. No caso do tripomastigota, a reatividade é menos pronunciada mostrando uma leve marcação no interior do parasita. / Alpha-L-fucose is a component of glycoproteins, inc1uding glycoproteins isolated from Tcruzi. a-L fucosidases have been isolated from different sources, but earlier studies were unable to detect this enzyme in T. cruzi epimastigotes (AVILA et al., 1979). In this work immunocytochemical and biochemical techniques have been used to localize and characterize a membrane-associated, neutral-pH-optimum alpha-L fucosidase from Trypanosoma cruzi epimastigotes. Light and electron microscopy specifically localized the alpha-L fucosidase on membranes in the posterior region of the epimastigotes and on the parasite surface. Immunoreactivity for alpha-L-fucosidase, a1though less intense, was also detected on the surface of trypomastigotes. Fractionation of epimastigotes homogenates indicated that over 50% of the a-Lfucosidase activity was associated with the 80 000 g pellet. This pellet-associated activity could be solubilized with 1 M NaCl or with 1% Triton X-I 00, suggesting that alpha-L-fucosidase is peripherally associated with membranes. Analysis of alpha-L-fucosidase on epimastigote extracts indicated that the enzyme had a pH-activity curve (with an optimum near 7) which was comparable to other alpha-L-fucosidases reported in the literature. A higher specific activity (in units/mg) was found in epimastigotes as compared to the other differentiation stages of the parasite: 0.028 for epimastigotes, 0.002 for metacyc1ic trypomastigotes and 0.015 for tissue - cultured trypomastigotes. SDS/PAGE and Westem blotting analysis indicated that epimastigotes have a protein band of 50 kDa which was immunoreactive with anti-alpha-L-fucosidase antibodies.