The Study With Fluorescent Probe TNP-AMP On Binding Sites Of Inhibitors To Cyanobacteria FBPase

Nowadays, China has been badly affected by cyanobacterial bloom. Cyanobacterial bloom will not only cause lethal effects to humans and aquatic ecosystems, but also pose serious risks for economic vitality of tourist industry. It is in urgent need of finding high-efficiency algaecide. To develop the high-efficiency inhibitors, a promising candidate target is needed. And it is also significant to study the mechanism of interaction between the enzyme and the inhibitors.Cyanobacteria Fructose-1,6-bisphosphatase(FBPase) plays a very important role in the Calvin cycle of photosynthesis of cyanobacteria, the content of which in cyanobacteria remains much lower than other enzyme. This makes Cyanobacteria FBPase a promising target for researching inhibitors.There are two kinds of binding sites of inhibitors to cyanobacteria FBPase:active site and allosteric site. Studying the binding sites of inhibitors to cyanobacteria FBPase is the very first step of studying the mechanism of interaction between the cyanobacteria FBPase and the inhibitors. Adding FBP, AMP and inhibitors to mixture of fluorescent probe TNP-AMP and cyanobacteria FBPase in a particular way and determining the fluorescence intensity, we established a way to predict the binding sites of inhibitors to cyanobacteria FBPase.The main work carried out to accomplish this thesis is as follows:1. The cyanobacteria FBPase was expressed in Escherichia coli, using pET-28a(+) as the expression plasmid. The enzyme was purified with affinity chromatography and analyzed by SDS-PAGE electrophoresis. And the yield of enzyme was 18.6 mg/L(LB medium).2. Established the system to determine the fluorescence intensity of fluorescent probe TNP-AMP Binding to cyanobacteria FBPase. The optimum temperature is 30℃ and the optimum pH is 8.0. The optimum final concentration of fluorescent probe TNP-AMP is 12.9μM and the optimum final concentration of cyanobacteria FBPase is 15μM. The the optimum final concentration of Mn2+is 400μM.3. Studied the fluorescence intensity of mixture of Mn2+and fluorescent probe TNP-AMP and the fluorescence intensity of mixture of Mn2+and cyanobacteria FBPase. Came to the conclusion that the reason of the enhancement of the fluorescence intensity, when Mn2+added to the mixture of fluorescent probe TNP-AMP and cyanobacteria FBPase, is that Mn2+will cause structure changing of the cyanobacteria FBPase, so that fluorescent probe TNP-AMP will bind more solidly to cyanobacteria FBPase.4. Added FBP, the binding site of which is known as active site, or AMP, the binding site of which is known as allosteric site, to the mixture of fluorescent probe TNP-AMP, cyanobacteria FBPase and Mn2+, and measured fluorescence quenching of this system. Came to the conclusion that fluorescent probe TNP-AMP binds not only to active site of cyanobacteria FBPase, but also to allosteric site.5. Added F6P, the binding site of which is known as active site, to the mixture of fluorescent probe TNP-AMP, cyanobacteria FBPase and Mn2+to validate the feasibility and accuracy of the way using fluorescent probe TNP-AMP to predict the binding sites of inhibitors to cyanobacteria FBPase.6. An inhibitors screening, taking the cyanobacteria FBPase as a target, has been made over three series of compounds including hs, hx and x. After analyzing the result, the measure of the half maximal inhibitory concentration(ICso) of 8 compounds from x series has been taken. Among them, x5 shows the lowest ICso, and the value is ICso=2.31±0.236μM.