Inhibition Kinetics And Inhibition Mechanism Of Compounds With Maleic Anhydride Structure Against Protein Phosphatases

The protein phosphatase is a kind of enzymes that remove a phosphate group from its substrate by hydrolysing phosphoric acid monoesters. It is the most common and important effectors in biological processes. Tautomycin as a compound with maleic anhydride presented high inhibitory activity against protein phosphatase. Due to the excellent biological activities, we can study the inhibition kinetics and inhibition mechanism of compounds with maleic anhydride structure against protein phosphatase. The natural compounds whether or not be used as protein phosphatase inhibitors in new biological pesticides.This article extracted protein phosphatase from Botrytis cinerea to research into a series of enzymatic properties. The purified phosphatase was used as a target to study kinds of maleic anhydride structure compounds inhibition.The protein phosphatase from B. cinerea was purified to30.69fold with a yield of39.26%through ultrasonication, centrifugation, ammonium sulfate precipitation and ion-exchange chromatography. The enzyme had only a single band on SDS-PAGE.Its optimum pH and temperature for reaction were pH4.0and37℃. The enzyme stable conditions were pH3.0～6.0and below50℃. A survey of effects of metal ions on the enzyme showed that the metal ions such as Fe2+, Ca2+, Ba2+, Mg2+activated the enzyme and Zn2+, Cu2+, Mn2+inhibited the enzyme. The Vmax and Km values of protein phosphatase forp-NPP were12.71mmol/L·min-1and12.84mmol/L.The inhibition effects of maleic anhydride on the protein phosphatase from B. cinerea were studied, tautomycin、4,5-dichlorophthalic anhydride、 phenylmaleic anhydride、dichlormaleic anhydride、tetrabromophthalic anhydride、dimethylmaleic anhydride and tetrachlorophthalic anhydride showed high inhibitory activity. Their IC50were0.57mmol/L、4.98mmol/L、5.85mmol/L、6.64mmol/L、8.43mmol/L,10.11mmol/L and14.5mmol/L, respectively. And the inhibition mechanism of tautomycin、dichlormaleic anhydride、tetrabromophthalic anhydride and tetrachlorophthalic anhydride were irriversible inhibition. The concentration and reaction time of compunds acted the effects. Dimethylmaleic anhydride and tautomycin inhibited the growth of B. cinerea.In sum, some of the compounds with maleic anhydride investigated in the study had good antifungal activity against B. cinerea as protein phosphatase inhibitors.