| Glycosidases are crucial in many biological processes, including breakdown of edible carbohydrates, eukaryotic glycoprotein processing, and polysaccharide and glycoconjugate anabolism and catabolism. Glycosidases are also involved in a variety of metabolic disorders and other diseases such as diabetes, gaucher disease, viral attachment, bacterial infection, and cancer formation. (3-Glucosidase is a glucosidase enzyme that acts uponβ1→4 bonds linking two glucose or glucose-substituted molecules. It catalyzes the hydrolysis of terminal non-reducing residues in beta-D-glucosides with release of glucose and plays an important role in maintaining normal physiology function of organisms.To prevent and treat those kinds of metabolic disease, it is high important to regulate and control enzyme activity in vivo. Thus, it is great desired to study the interactions between suitable inhibitors and target enzyme. In this thesis, the fluorescence spectroscopy, UV-visible absorption spectroscopy and CD spectroscopy were applied to investigate the interactions between puerarin, quercetin, luteolin andβ-glucosidase, respectively. Molecular docking, which plays an important role in rational drug design, is also applied to study the molecular basis involved in interaction between ligand and receptor. This thesis consists of four chapters as followings.In chapter one, the current research activities on the interaction between small drug compounds and biological receptor were summarized. Then, the objectives and methods of this paper were outlined.In chapter two, the spectrofluorimetry was applied to investigate the interaction between puerarin andβ-glucosidase in combination with molecular simulation. The binding reaction was simultaneously studied by the AutoDock 4.2 molecular docking model. The interaction between puerarin andβ-glucosidase was studied using fluorescence quenching method and synchronous fluorimetry,3D fluorimetry and CD spectroscopy. Data from fluorescence spectroscopy indicated that these interactions result in the endogenous fluorescence quenching ofβ-glucosidase, which belongs to a static quenching mechanism, and fluorescence energy transfer occured between puerarin andβ-glucosidase. The calculated binding constants were 2.43×104 L·mol-1,1.87×104 L·mol-1 and 1.03×104 L·mol-1 at 290K,300K and 310K, respectively. The evidences from synchronous fluorescence showed the effect of puerarin on the microenvironment aroundβ-glucosidase in aqueous solution. Molecular docking was performed to reveal the possible binding mode or mechanism and suggested that puerarin can bind strongly toβ-glucosidase. The results revealed that puerarin tended to bind withβ-glucosidase mainly by hydrogen bond and van der Waals'forces.In chapter three, combined with molecular docking model, a fluorescence method was applied to investigate the interaction between quercetin and P-glucosidase and the acting mechanism. The interaction between (3-glucosidase and quercetin, as well as the enzyme inhibitor 4-nitrophenyl-(3-D-thioglucoside, was studied by the AutoDock4.2 molecular docking model, respectively. The binding reaction was simultaneously studied using fluorescence quenching method. The results showed that these interactions result in the endogenous fluorescence quenching of (3-glucosidase, which belongs to a static quenching mechanism. The calculated binding constants were 4.36×104 L·mol-1,4.04×104 L·mol-1 and 3.18×104 L·mol-1 at 290K,300K and 310K, respectively. The results revealed that quercetin tended to bind withβ-glucosidase mainly by hydrogen bond and hydrophobic interaction, as well as electrostatic forces. The inhibition test showed that the activity ofβ-glucosidase can be inhibited by quercetin. The determined bimolecular rate constant (ki) was 4.46×103 (mol·L-1)-1·min-1. Both fluorescence spectroscopy and molecular docking are complimentary to each other for the investigation of the interaction betweenβ-glucosidase and quercetin from the experimental and theoretical view.In chapter four, the spectrofluorimetry was applied to investigate the interaction between luteolin andβ-glucosidase in combination with molecular simulation. The interaction between luteolin andβ-glucosidase was studied using fluorimetry and synchronous fluorimetry 3D fluorescence spectroscopy and CD spectroscopy. Data from fluorescence spectroscopy indicated that these interactions result in the endogenous fluorescence quenching ofβ-glucosidase, which belongs to a static quenching mechanism. The calculated binding constants were 2.38×105 L·mol-1,1.03×105 L·mol-1 and 0.60×105 L·mol-1 at 290K,300K and 310K, respectively. The evidences from synchronous fluorescence showed the effect of luteolin on the microenvironment aroundβ-glucosidase in aqueous solution. Molecular docking was performed to reveal the possible binding mode or mechanism and suggested that luteolin can bind strongly toβ-glucosidase. The inhibition test showed that the activity ofβ-glucosidase can be inhibited by luteolin. The determined bimolecular rate constant (ki) was 6.26×102 (mol·L-1)-1·min-1. The binding reaction was simultaneously studied by the AutoDock 4.2 molecular docking model. The results revealed that luteolin tended to bind withβ-glucosidase mainly by hydrogen bond and van der Waals'forces. |
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