碩士 / 國立臺灣大學 / 生化科學研究所 / 93 / alpha-L-Fucosidases are exo-glycosidases to cleave alpha-linked L-fucose residues from the glycoconjugates that usually participate in a variety of important biological processes. Decreased α-L-fucosidase activity is related to a number of pathological conditions such as inflammation, cancer, cystic fibrosis and fucosidosis. Therefore, discovery of potent and selective inhibitors certainly sheds light on the study of their functions and development of therapeutic agents.
Previously, our lab established rapid synthesis of fuconojirimycin-based inhibitors in microtiter plates for high-throughput screening, which rapidly identified potent and selective inhibitors against alpha-L-fucosidases from Thermotoga maritima and human tissue. Interestingly, slow, tight-binding inhibition was found in the study of the former enzyme, distinct from the reversible inhibition of the latter. These molecules thus have higher affinity with the Thermotoga maritima -L-fucosidases than that with the human enzyme, with the best inhibitor exhibiting inhibitory difference up to 5,400-fold .
According to the homology modeling and molecular docking, the drastic difference in the inhibition was probably attributed to the hydrophobic aglycon binding site that was absent in the human fucosidase. To decipher the interesting distinction at molecular level, six residues (W58, F59, Y64, L191, M225 and Y267) likely involved in the aglycon binding were changed to alanine by site-directed mutagenesis. Slow-binding inhibition was diminished to different degrees in activity assays of these mutants. Futhermore, intrinsic fluorescence analysis indicated that Y64A and Y267A mutants had no significant change upon inhibitor titration and thus behaved like their human counterpart. The result is not only consistent with the corresponding kinetic data, but also corroborates the role of proposed hydrophobic residues in the aglycon binding site.
| Identifer | oai:union.ndltd.org:TW/093NTU00103006 |
| Date | January 2005 |
| Creators | Yu-Nong Lin, 林宥穠 |
| Contributors | Chun-Hung Lin, 林俊宏 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 93 |
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