The Inhibitory Effect Of HSYA On ?-glucosidase Was Studied From The Perspective Of Enzyme Kinetics And Molecular Simulation

Objective?-glucosidase is a kind of enzyme closely related to type 2 diabetes.It ean specifieally hydrolyze alpha-1,4 glycosidic bond of ?-glucose to release glucose which is harmful to the diabetic patients.Inhibiting the activity of ?-glucosidase is an effective way to control postprandial blood glucose elevation,which can reduce production and absorption of monosaccharide.Thus,research of inhibitors of ?-glucosidase is of great significance for treatment of diabetes and avoiding the occurrence of complications.At present there are acarbose,miglitol and other ?-glucosidase inhibitors applied in clinical.However,all of the inhibitors have certain side effects which makes it important to find cheap ?-glucosidase inhibitors of low toxicity from Chinese traditional medicine.HSYA is the effective component of saffron.It has four benzene and phenol hydroxyl groups in the structure and it belongs to the flavonoids.The aim of this study is to do research on the inhibitory effect of HSYA on ?-glucosidase,so that it can lay the foundation for the development of ?-glucosidase inhibitor.Method1.The inhibitory effects of HSYA of different concentrations on the activity of?-glucosidase:The ultraviolet spectrophotometry was used in this experiment.Under the condition of 37?,pNP was formed in pNPG hydrolysis reaction with ?-glucosidase.At this time,the change rate of absorbance per minute which was measured at 400 nm is defined as the activity of ?-glucosidase.2.Study whether the inhibition is reversible by reaction of different concentrations of HSYA and different concentrations of ?-glucosidase:Under the condition of fixed substrate concentration,HSYA of different concentrations and different concentrations of enzyme were incubated.The activity of enzyme was detected in the substrate system with the addition of the incubation products into it.Based on the measured data,the relationship between enzyme activity and the concentration of the relationship was plotted.So that it is possible to judge whether the inhibition effect of HSYA on ?-glucosidase is reversible according to the relationship.3.Study the type of inhibition by the reaction of ?-glucosidase inhibited by different concentrations of HSYA and different concentrations of substrate:Under the condition of fixed concentration of enzyme,the activity of ?-glucosidase was measured by the reaction of different concentrations of HSYA and different concentrations of substrate.According to the measured data and Lineweaver-Burk double reciprocal plot,the inhibition kinetics curves of a-glucosidase were drawn and the type of inhibition effect of HSYA on ?-glucosidase was determined.4.Study the effect of HSYA on the structure of ?-glucosidase by reaction of different concentrations of HSYA and different concentrations of ?-glucosidase:A certain concentration of ?-glucosidase was mixed with different concentrations of HSYA,the mixture was incubated at 25 ? for 2 hours.The changes of endogenous fluorescence and fluorescence of ANS were measured by fluorescence spectrophotometer.5.Study on the inhibition mechanism of HSYA on ?-glucosidase by homology modeling and molecular dynamics.Based on the protein sequences available in the database,the PQR-SA program was used to model the structure of alpha.The structure is generated as the initial structure of docking and molecular dynamics simulation.The active site of 3D model was predicted through the combination of ?-glucosidase ligand and ?-glucosidase.The ligand HSYA was predicted using Marvin software.The structure of ?-glucosidase and HSYA predicted were studied using molecular docking simulation.In this way,the binding method and binding mechanism could be understood.Result1.3mM of HSYA can completely inhibit the activity of luM alpha-glucosidase2.The inhibition of HSYA on alpha-glucosidase is competitive and reversible3.HSYA can affect the tertiary structure of alpha-glucosidase,and expose hydrophobic groups of alpha-glucosidase4.Molecular simulation analysis showed that HSYA can bind with amino acid residues near the activity center through its benzene ring structure,so that it affects the combination of alpha-glucosidase enzyme with its substrate,and inhibits the activity of alpha-glucosidase.ConclusionHSYA can inhibit the activity of alpha-glucosidase in concentration dependent,reversible,competitive manner.The inhibition is related to the fact that HSYA can change the tertiary structure and expose hydrophobic groups of alpha-glucosidase as well as the fact that HSYA can bind the amino acid residues of alpha-glucosidase activity sites via its four benzene rings.This conclusion provides a theoretical basis for the potential of HSYA as a potential inhibitor and it also lay the foundation that HSYA may become an effective medicine for the clinical treatment of diabetic patients with postprandial hyperglycemia.