Study On Enzymatic Properties And Catalytic Reaction Characteristics Of Acetylhydroxyacid Synthase Catalytic Subunit Of Mycobacterium Tuberculosis

Acetohydroxy acid synthase(AHAS)is the first common enzyme in the biosynthetic pathway of branched-chain amino acids in plants and microorganisms.Since this pathway does not exist in humans and other mammals,AHAS is a very promising target for screening herbicides and antibiotics.MostAHAS from bacteria or fungi are heterotetramers,composed of two large subunits and two small subunits.The large subunit mainly plays a catalytic role,so it is called the catalytic subunit(CSU);The small subunit mainly plays a regulatory role,so it is called regulatory subunits(RSU).The CSU of AHAS also has full catalytic activity when it exists alone.In this thesis,AHAS of Mycobacterium tuberculosis origin has investigated.First,the engineered bacterial strain used to express the recombinant MtAHAS-CSU was prepared,and the recombinant protein MtAHAS-CSU was obtained in high purity and good activity by using an automatic induction expression protocol and affinity chromatography.Then the properties of the enzyme were characterized.The results show that the optimal temperature of MtAHAS-CSU is 37°C;the optimal reaction p H is 7.5,and the effect is better when KH₂PO₄is used as a buffer;The proper reaction time is 30 minutes and prolonged reaction time might cause product inhibition.In the mean time,the creatine-naphthol method were used to determine the K_m^Pyrvalues of MtAHAS-CSU for pyruvate,and the result was 4.5 m M.In addition,the influences of various divalent metal ions and cosolvents on enzyme activity were also analyzed.The influences of commercial herbicides Chlorsulfuron and Chlorsulfuron on MtAHAS-CSU were also investigated.The results showed that both herbicides had a strong inhibitory effect on mtahas CSU activity.The concentrations of Chlorsulfuron methyl and Chlorsulfuron methyl inhibited 90%enzyme activity were 15 m and 0.25 m,respectively.This study also tested the effect of 16 kinds of volatile oil of traditional Chinese medicines on the activity of MtAHAS-CSU preliminarily.The results showed that the volatile oil of Schisandra chinensis has a certain degree of inhibitory effect on the enzyme activity(at a concentration of 1 mg/m L,80%of the enzyme activity is inhibited).AHAS has two natural substrates,pyruvate and 2-ketobutyrate.At present,there have been many studies on the AHAS catalyzed reaction using pyruvate as a substrate.However,the studies on the prodct formation and distribution of the AHAS reaction empolying pyruvate and 2-ketobutyrate as substrates are still on early stage.In this study,4-nitro-o-phenylenediamine and 2,4-dinitrophenylhydrazine were used as derivatizing reagents,respectively in a pre-column derivatization-HPLC method to qualitatively and quantitatively analyze the substrate consumption and product formation and distribution of the single-substrate reaction and the double-substrate reaction catalyzed by MtAHAS-CSU.The determination of the AHAS-catalyzed single-substrate reaction,showed that the main product of the reaction,acetolactate as determined in its decarboxylated form acetoin was produced with a yield of up to 82.6%,and 2,3-butanedione with a yield of 16.2%.Meanwhile,the measurments also indicated the consumption rate of the substrate pyruvate was as high as99.6%.From these data we can see that the consumption of the substrate matches well the formation of the products.As for the dual-substrate reaction,after decarboxylation,nine products were detected:2,3-butanedione(14.8%),2,3-pentanedione(31.2%),acetoin(14.6%),2-hydroxy-3-pentanone(4.6%),3-hydroxy-2-pentanone(15.42%),4-hydroxy-3-hexanone(11.44%),3,4-hexanedione(2.12%),acetaldehyde(0.61%),propionaldehyde(2.25%).The research in this article provides a basis for the following research on the catalytic mechanism of MtAHAS-CSU,and also provides a reference for the in-depth study of the catalytic mechanism of different types of AHAS.It also has certain reference significance for screening antibacterial lead compounds with MtAHAS as a target.