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1.
Summary.  This paper addresses the existence and role of a catalytic nucleophile in retaining glycoside hydrolases and retaining glycosyl transferases. Although the former has now been established beyond doubt, such is not the case with the latter. We report reliable procedures for the synthesis of various 2-deoxy-2-fluoro glycosyl nucleoside diphosphates, useful donor analogues for the study of the mechanism of action of retaining glycosyl transferases. Received October 16, 2001. Accepted November 7, 2001  相似文献
2.
Catalytically efficient β-d-xylosidase from Selenomonas ruminantium (SXA) exhibits pK as 5 and 7 (assigned to catalytic base, D14, and catalytic acid, E186) for k cat/K m with substrates 1,4-β-d-xylobiose (X2) and 1,4-β-d-xylotriose (X3). Catalytically inactive, dianionic SXA (D14E186) has threefold lower affinity than catalytically active, monoanionic SXA (D14E186H) for X2 and X3, whereas D14E186 has twofold higher affinity than D14E186H for 4-nitrophenyl-β-d-xylopyranoside (4NPX), and D14E186 has no affinity for 4-nitrophenyl-α-l-arabinofuranoside. Anomeric isomers, α-d-xylose and β-d-xylose, have similar affinity for SXA. 4-Nitrophenol competitively inhibits SXA-catalyzed hydrolysis of 4NPX. SXA steady-state kinetic parameters account for complete progress curves of SXA-catalyzed hydrolysis reactions. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.  相似文献
3.
4.
A novel thermostable β-galactosidase gene, designated as GkGallA, from the thermophilic bacterium Geobacillus kaustophilus HTA426 was cloned and heterologously overexpressed in Escherichia coli(E, coli). Based on the sequence analysis, GkGallA belongs to the glycosyl hydrolase family 1 that was the first β-galactosidase of bacterial origins expressed by us in this family. The apparent molecular weight of GkGallA determined by sodium deodecyl sulfate-polyacrylamide gel electrophoresis is 52000. It exhibited the highest activity toward p-nitrophenyl-β-D-galactopyranoside at pH 7.8 and 70℃ and displayed high thermal stability, Divalent cations are prerequisite for the activity of GKGallA, with the highest activity in the presence of Mn2+. Moreover, the three-dimensional structure of GkGaI1A was modeled to speculate the structure of the catalytic residues and the reac- tion mechanism. The catalytic residues consisting of Glu166 and Glu355 were verified by site-directed mutagenesis.  相似文献
5.
Fragment‐based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small‐molecule enzyme activators. Herein, we describe the discovery and characterization of small‐molecule activators of a glycoside hydrolase (a bacterial O‐GlcNAc hydrolase). A ligand‐observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90 % increase in kcat/KM values (kcat=catalytic rate constant; KM=Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both KM and Vmax values (Vmax=maximum rate of reaction). The compounds appear to stabilize an active “closed” form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in vivo, and could also be used for glycoside hydrolase activation in many industrial processes.  相似文献
6.
α‐l ‐Fucosidases (EC 3.2.1.51) are exo‐glycosidases. On the basis of the multi‐alignment of amino acid sequence, α‐l ‐fucosidases were classified into two families of glycoside hydrolases, GH‐29 and GH‐95. They are responsible for the removal of l ‐fucosyl residues from the non‐reducing end of glycoconjugates. Deficiency of α‐l ‐fucosidase results in Fucosidosis due to the accumulation of fucose‐containing glycolipids, glycoproteins and oligosaccharides in various tissues. Recent studies discovered that the fucosylation levels are increased on the membrane surfaces of many carcinomas, indicating the biological function of α‐l ‐fucosidases may relate to this abnormal cell physiology. Although the gene of human α‐l ‐fucosidase (h‐fuc) was cloned, the recombinant enzyme has rarely been overexpressed as a soluble and active from. We report herein that, with carefully control on the growing condition, an active human α‐l ‐fucosidases (h‐Fuc) was successfully expressed in Escherichia coli for the first time. After a series steps of ion‐exchange and gel‐filtration chromatographic purification, the recombinant h‐Fuc with 95% homogeneity was obtained. The molecular weight of the enzyme was analyzed by SDS‐PAGE (∼50 kDa) and confirmed by ESI mass (50895 Da). The recombinant h‐Fuc was stable up to 55 °C with incubation at pH 6.8 for 2 h; the optimum temperature for h‐Fuc is approximately 55 °C. The enzyme was stable at pH 2.5–7.0 for 2 h; the enzyme activity decreased greatly for pH greater than 8.0 or less than 2.0. The Km and kcat values of the recombinant h‐Fuc (at pH 6.8) were determined to be 0.28 mM and 17.1 s−1, respectively. The study of pH‐dependent activity showed that the recombinant enzyme exhibited optimum activity at two regions near at pH 4.5 and pH 6.5. These features of the recombinant h‐Fuc are comparable to the native enzyme purified directly from human liver. Studies on the transfucosylation and common intermediate of the enzymatic reaction by NMR support that h‐Fuc functions as a retaining enzyme catalyzing the hydrolysis of substrate via a two‐step, double displacement mechanism.  相似文献
7.
β-d-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k cat, k cat/K m) for promoting hydrolysis of 1,4-β-d-xylooligosaccharides. 1H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 1,4-β-d-xylobiose (X2). Progress curves of 4-nitrophenyl-β-d-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k cat and k cat/K m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites −1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites bey ond−1 are blocked by protein and subsites bey ond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k cat and k cat/K m by <103 and to asimilar extent for substrates 4NPX and 4-nitrophenyl-α-l-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs k cat/K m with pK a values of 5.0 and 7.0 assigned to D14 and E186, respectively. k cat (4NPX) has a pK a value of 7.0 and k cat (4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, “dianionic” enzyme form (D14-E187-) binds 4NPX but not 4NPA. The mention of firm names or trade products does not imply that they are end orsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.  相似文献
8.
Glycoside hydrolases (GHs) have attracted considerable attention as targets for therapeutic agents, and thus mechanism‐based inhibitors are of great interest. We report the first structural analysis of a carbocyclic mechanism‐based GH inactivator, the results of which show that the two Michaelis complexes are in 2H3 conformations. We also report the synthesis and reactivity of a fluorinated analogue and the structure of its covalently linked intermediate (flattened 2H3 half‐chair). We conclude that these inactivator reactions mainly involve motion of the pseudo‐anomeric carbon atom, knowledge that should stimulate the design of new transition‐state analogues for use as chemical biology tools.  相似文献
9.
10.
Zhang X  Liu N  Yang F  Li J  Wang L  Chen G  Gao P 《Electrophoresis》2012,33(2):280-287
Based on digital image analysis techniques and a series of optimizations in native electrophoresis, a new direct method to simultaneously detect the intrinsic properties of each active component in the enzymatic system of glycoside hydrolase was established. The key technique is that the concentration changes of substrate (or product) on the gel can be determined quantitatively by the gray value changes of the corresponding band after electrophoretic separation. In this manner, the catalytic characteristics of each glycoside hydrolase component were demonstrated in situ and were easily determined after immersing the gel in a series of solutions containing substrates or their derivatives. Because of its high throughput, great sensitivity, and convenient operation, this method can be used to demonstrate the natural diversity of glycoside hydrolases and to study spatial and temporal regulation in multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of glycoside hydrolases.  相似文献
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