Kinetic And Thermodynamic Analysis Of Enzymatic Reactions Of Four Sesquiterpene Synthase

The sesquiterpene synthase-catalyzed reaction starts with the cleavage of the diphosphate moiety by farnesyl pyrophosphate,resulting in unstable terpene carbocations.Subsequently,the catalytic profile of the substrate binding site in sesquiterpene synthase initiates and directs a cascade of carbocation reactions(eg,double bond migration,hydride and methyl transfer or deprotonation),resulting in a single terpene or multiple of terpenes.Studies on the mutation of sesquiterpene synthase can identify the effect of protein amino acid sequence on the catalytic efficiency and product specificity of the enzyme,further elucidate the catalytic mechanism of sesquiterpene synthase,and Can provide theoretical guidance for the development of new prodrugs in practical applications.There is a rate-limiting step in the catalytic mechanism of sesquiterpene synthase.One case study found that the isomerization process of(E,E)-FPP to NPP is to limit the rate of chemical catalysis in the catalytic reaction pathway of Trichodene synthase(FSTS).It has been reported that theoretical calculations found that the process of isomerization of(E,E)-FPP to transoid(R,E)-NPP is an endothermic reaction.The catalytic activity and kinetic behavior of enzymes are very important in biochemical and biotechnology research.At present,there are few studies on the rate-limiting steps of sesquiterpene synthase,and clear rate-limiting steps can guide site-directed mutations and improve the catalytic activity of enzymes.Isot hermal titration calorimetry method can be used to measure enzyme kinetic parameters and thermodynamic parameters.Because the thermal energy generated as the reaction progresses is a direct and sensitive observable event.The principle of this method is to determine the kinetic and thermodynamic behavior of the enzymatic reaction by recording the change in the endothermic/exothermic chemical reaction over time through chemical titration experiments of enzymes and substrates.In order to systematically study the catalytic reaction kinetics and thermodynamic characteristics of sesquiterpene synthase,In this project,four representative catalytic reactions of sesquiterpene synthase,AaADS,LdBOS,ZoBS,and ZmTPS4,are used as research objects.The catalytic reaction kinetics and thermodynamics are analyzed,and the characteristics of sesquiterpene synthase catalytic mechanism are discussed.The motif was subjected to point mutation to further explore the role of the RXR motif in the catalytic mechanism of sesquiterpene synthase.The following experimental results were obtained.After the first R mutation in the RXR motif of AaADS,LdBOS,ZoBS,and ZmTPS4 was changed to K,the types or contents of terpenols produced by(E,E)-FPP catalyzed by four sesquiterpene synthase enzymes increased;After AaADS-R262 A catalyzed(E,E)-FPP,no product was formed.After LdBOS-R260 A,ZoBS-R266 A,and ZmTPS4-R271 A catalyze(E,E)-FPP,the amount of product is greatly reduced,and the enzyme activity is weakened.Both ZOBS and ZmTPS4 catalyzed(E,E)-FPP and(S,E)-NPP had negative enthalpy changes,and the absolute enthalpy change of the former was greater than the latter.Compared with the time required by ZoBS and ZmTPS4 to catalyze(S,E)-NPP to produce a molecular product,the time required by ZoBS and ZmTPS4 to catalyze(E,E)-FPP to produce a molecular product is longer.From the analysis of the characteristics of enzymes and the relationship between enzymes and substrates,the following conclusions are drawn:1 Kinetic studies indicate that the process of sesquiterpene synthase catalyzing the conversion of FPP to NPP is a step that limits the rate of chemical catalysis in the course of the enzyme-catalyzed reaction;2 Thermodynamic studies show that sesquiterpene synthase catalyzes the production of heat from FPP to produce products,and the enthalpy change value of FPP to NPP is negative.It is found that the process of FPP to NPP conversion is exothermic.