The Study Base On The Type Ⅱ Fructose-1,6-bisphosphatase GlpX From Escherichia Coli And The Screening Of Its Inhibitors

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol.Fructose1,6-bisphosphatase (FBPase) is a key enzyme for gluconeogenesis, the enzyme catalyzed a reversible reaction, and the enzyme catalyzed hydrolysis of fructose1,6-bisphosphate (FBP) to fructose6-phosphate (F6P)and phosphate (Pi). It is the inverse reaction of fructose phosphate kinase catalytic glycolysis,the product fructose-6phosphate is an important in various biosynthetic pathway precursors.In order to comprehensively understanding of various FBPase, Our group began to study human FBPase and Escherichia coli FBPase-GlpX. The human FBPase do enzyme target for the treatment of diabetes,and study this enzyme E. coli FBPase-GlpX meaningful for understanding bacterial FBPase. In addition,we will have a better understanding of the characteristics of the higher plants, animals, cyanobacteria and bacteria FBPase,and which uniformity and differences, including biochemical characterization, spatial structure, the active cavity, combination and inhibitors and so on.the specific contents are as follows:Firstly, by the protein purification methods, we purified the a higher purity coli FBPase-GlpX protein and the protein size of36kDa.Secondly, we improved the activity conditions of E. coli the the FBPase-GlpX and measured the activity of GlpX. Through research of biochemical characteristics for GlpX,we obtained the activity conditions, the activity conditions ara as follows:protein content is2.1mg/ml, the activity of GlpX is2.0±0.3U/mg, final concentration of Mn2+50μM, final concentration of FBP0.5mM, pH8.0, reaction time is5min, the reaction temperature is30℃.Finally, we screened more then200series of compounds in enzyme levels perserved in our laboratory, including23series compounds of A,30series compounds of C,17series compounds of D7S D13S,30series compounds of H,14series compound of S,34series compoundof LH,110series compounds TQD.The inhibition rate is more than80%have12series compounds,and more than50%have34. The TQD series of compounds have high inhibitory rate of more than80%in the coarse screening, and measured their IC50,and we found in these10compounds TQD152, TQD151,TQD154and TQD153have higher inhibition effect,and which IC50are respectively3.40±1.83μM,3.91±1.45μM,7.18±1.84μM,7.87±1.84μM,all under10μM.