Theoretical Studies On The Mechanisms Of Several Enzymes

Enzymes have many advantages:remarkable catalytic activity,high specificity,mild reaction conditions,et al.A thorough study of the catalytic mechanism of enzymes will help us to understand the biological function of enzymes,and provide a theoretical basis for the modification and application of enzymes.The structure of enzymes,kinetics and mutation mechanisms have been obtained through a large number of experiments.However,many problems are still unclear,as a result of the complexity of enzymes.For instance,the structures of intermediate and transition involved in the reaction will not be characterized.as well as the elementary reactions,the rate-determining step,and the energetic information.The computational method can be applied to explore the catalytic reaction at the molecular level,which can be mutually verified and complemented with a large number of experimental data.In recent years,the computational enzymology has been developed.The computational results have been used to explain experiments by researchers,which will be further guiding the experiment.With the development of computer software and the improvement of theoretical calculation methods,computational chemistry has been widely used in the study of large-scale biochemical systems.It has been an important tool for investigating the mechanism of enzymes.In this dissertation,we employed the combined quantum mechanics/molecular mechanics(QM/MM)method to systematically explore the catalytic mechanisms of several important cofactor-containing enzymes.According to the QM/MM calculations,we analyzed the interaction between the enzyme and substrate;investigated the mechanism of coenzyme factors(metal iron ions,flavin and PLP);captured the details of enzymatic reaction,the structures of intermediate and transition involved in the reaction,energetic information;described the electron transfer process;and identified the important role of some residues.These results are of great significance for further understanding these enzymes,and provide useful theoretical direction for application of other related enzymes.The main research works of this dissertation are as followed:(1)A QM/MM study of the catalytic mechanism of oxidative decarboxylase(HemQ)The peroxide dependent coproheme decarboxylase HemQ from Geobacillus stearothermophilus catalyzes two key steps in the synthesis of heme b,i.e.,two sequential oxidative decarboxylation of coproporphyrinogen Ⅲ(coproheme Ⅲ)at propionate groups P2 and P4.In the binding site of coproheme Ⅲ,P2 and P4 are anchored by different residues(Tyrl44,Arg217 and Ser222 for P2,Tyr113,Lysl48 and Trp156 for P4),however,strong experimental evidence supports that the generated Tyr144 radical acts as the unique intermediary for hydrogen atom transfer(HAT)from both reactive propionates.So far,the reaction details are still unclear.Herein,we carried out quantum mechanics/molecular mechanics calculations on the full structure of HemQ enzyme to explore the decarboxylation mechanism of coproheme Ⅲ.In our calculations,coproheme Cpa I,the Fe(Ⅳ)=O coupled to a porphyrin radical cation(por+)with four propionate groups,was used as the reactant model.Our calculations reveal that Tyr144 is directly involved in the decarboxylation of propionate group P2.Firstly,the proton-coupled electron transfer(PCET)occurs from Tyrl44 to P2,generating the Tyrl44 radical,which then abstracts a hydrogen atom from the Cβ of P2.It is the porphyrin radical cation(por+)that makes the PCET from Tyr144 to P2 to initiate the decarboxylation of P2.Finally,the electron transfers from the Cβ through the porphyrin to the iron center,leading to the generation of the vinyl and CO2.Importantly,the decarboxylation of P4 mediated by the lysine residue is calculated to be very difficult,which suggests that,after the decarboxylation of P2,the generated harderoheme III intermediate should re-bind in the active site where the propionate P4 occupies the binding site of P2,and Tyr144 again mediates the decarboxylation of P4.Thus,our calculations support the fact that Tyr144 is responsible for the decarboxylation of both P2 and P4.(2)Mechanistic insights into the ferulic acid decarboxylase(FDC)Ubiquinone plays a pivotal role in the aerobic cellular respiratory electron transport chain,whereas ferulic acid decarboxylase(FDC)is involved in the biosynthesis of ubiquinone precursor.Recently,the complete crystal structure of FDC(based on the co-expression of the A.niger fdcl gene in E.coli with the associated ubix gene from E.coli)at high resolution was reported.Herein,the detailed catalytic non-oxidative decarboxylation mechanism of FDC has been investigated by a combined quantum mechanics/molecular mechanics(QM/MM)approach.Calculation results indicate that,after the 1,3-dipolar cycloaddition of the substrate and cofactor,the carboxylic group can readily split off from the adduct,and the overall energy barrier of the whole catalytic reaction is 23.5 kcal/mol.According to the energy barrier analysis,the protonation step is rate-limiting.The conserved protonated Glu282 is suggested to be the proton donor through a "water bridge".Besides,two cases,that is,the generated CO2 escapes from the active site or remains in the active site,were considered.It was found that the prolonged leaving of CO2 can facilitate the protonation of the intermediate.In particular,our calculations shed light on the detailed function of both cofactors prFMNiminium and prFMNketamine in the decarboxylation step.The cofactor prFMNiminium is the catalytically relevant species compared with prFMNketamine.(3)Insights into the catalytic mechanism of the ergothioneine synthase(EgtB)Ergothioneine synthase(EgtB)is a unique non-heme mononuclear iron enzyme that catalyzes the sulfoxidation and C-S bond formation between γ-glutamyl cysteine(γGC)and N-a-trimethyl histidine(TMH)as a pivotal step in the ergothioneine biosynthesis.A controversy has arisen regarding the sequence of sulfoxidation and C-S bond formation in the catalytic cycle.To clarify this issue,the QM/MM approach has been employed to investigate the detailed mechanism of EgtB.Two binding modes of O2 to Fe(II)("end-on"and "side-on")have been identified.Within the present computational model,the end-on binding mode of O2 is preferred.The open-shell singlet is calculated to be the ground state,whereas the quintet is the most active state.Moreover,the sulfoxidation is prior to the formation of the C-S bond,and the reaction mainly occurs on the quintet state surface.Due to the electron transfer from the yGC to the ferric superoxide,the sulfur atom of yGC has partial radical characteristics,which facilitates the attack of the distal oxygen atom on the sulfur radical of yGC to form the sulfoxide.The formation of TMH C2 anion,i.e.,the ion of the proton from the imidazole group in TMH by the Fe(IV)-oxo species,is the prerequisite for C-S bond formation,which is the rate-limiting step with an energy barrier of 21.7 kcal/mol.In addition,it is also found that although the resulting iron(Ⅲ)-oxo can easily abstract a proton from Tyr377 to generate a phenolic hydroxyl anion,the subsequent proton transfer from C2 to Tyr377 is calculated to be difficult;thus,Tyr377 is not directly involved in the sulfoxidation and C-S bond formation.Our calculations also reveal that the side-on mode is not the catalytically relevant species.This work provides a direct comparison with previous experimental and theoretical studies,which is helpful for understanding the catalysis of ergothioneine synthase and related enzymes.(4)Theoretical insights into catalytic mechanism of sulfoxide lyase(Egt2)Ergothioneine synthase Egt2 is a PLP-dependent enzyme,which is response for the C-S bond cleavage of sulfoxide.Egt2 plays an essential role in the last step of ergothioneine biosynthesis pathway.Although abundant information concerning the catalysis of Egt2 has been derived from the experiments,open questions still remain.For example,the structures and energetics of transition states and intermediates are still unknown;the role of conserved residue is unclear;and the catalytic reaction details need to be clarified.In 2018,Zhang et al.characterized the structural of PLP-dependent C-S lyase Egt2 and proposed the catalytic mechanism.It was found that the active site of wild-type Egt2,located at the interface of the two dimers,and the pyridine ring of PLP aligns parallel to the aromatic ring of Tyr134 via π-π stacking interaction.On the basis of the recently obtained crystal structures,QM/MM calculations have been carried out on the mechanism of C-S bond cleavage of sulfoxide.Calculation results indicate that,the deprotonated residue Tyr134 abstracts a proton from Ca to initiate the reaction.Then,the sulfoxide abstracts a proton from Tyr134 after isomerization,meanwhile,the C-S bond cleaves to form sulfinic acid intermediate and PLP-aminoacrylic acid intermediate.Subsquently,Lys247 attacks the C=N bond of PLP-aminoacrylic acid,leading to the formation of aminoacrylic acid and internal aldimine.The deprotonated residue Tyr134 acts as bases to accept a proton from Ca,and returns the proton back to sulfoxide group.These results shed light on the mechanism of Egt2 and provide a new perspective for the mechanism study of ergothioneine biosynthesis.