Theoretical Insights Into Catalytic Mechanisms Of Several Heme And Non-Heme Enzymes

Enzymes are a class of macromolecular biocatalysts,which could greatly accelerate the rate of chemical reactions in life,so that the substances and energy produced by metabolism can meet the needs of organisms.Compared with those non-biological catalysts,bioenzyme catalysts have a lot of advantages,such as the ability to react at room temperature and atmospheric pressure,the high reaction rate,the specificity of reactions,and the low price.However,because of the complexity of the enzymatic reactions,it is difficult to obtain the structures of the reaction intermediates and transition states,along with the energetic information of each step only by experimental methods.In recent years,computational chemistry has provided a powerful means to understand mechanisms of enzymatic reactions and the design and applications of biocatalysts.Among them,the combined quantum mechanics with molecular mechanics(QM/MM)has been more and more powerful for macromolecular systems.It combines both the accuracy of quantum mechanics and the efficiency of molecular mechanics.The QM/MM method and density functional theory(DFT)method were used to investigate the catalytic reaction mechanisms of non-heme dioxygenase ChaP,engineered evolutionary enzyme RA95.5-8F and the model compound iron porphyrin nitrene based on cytochrome P450 enzyme.The micro-mechanisms of the reactions were clarified at the atomic level.Besides,the structures,energy and structure-activity of the intermediates and transition states involved in the reactions were given.These computational results have provided a further supplement to the experiments and have theoretical significance for the mechanisms of enzymatic reactions and design of biocatalysts with high reactivity.The main research work of this thesis is as followed:(1)Theoretical study of iron porphyrin nitrene:formation mechanism,electronic nature and intermolecular C-H aminationIron-porphyrin compounds are the analogues of catalytic center of cytochrome P450 monooxygenase,and they have been one of hot topics because of their good catalytic performance.The formation mechanism and electronic structures of iron porphyrin nitrene intermediates,as well as the nitrene-mediated intermolecular C-H amination have been studied by performing DFT and ab initio complete active space self-consistent field(CASSCF)calculations.Compared with that of cobalt porphyrin nitrene and iron porphyrin carbene,the formation of iron porphyrin nitrene shows similar but different characteristics.Their formation all requires to undergo the "far" or "close"complexes,but these complexes correspond to different energies relative to their respective reactants(isolated metalloporphyrins and azides).The overall free energy barrier for the formation of iron porphyrin nitrene was calculated to be 10.6 kcal/mol on triplet state surface,which is lower than those of cobalt porphyrin nitrene and iron porphyrin carbene.The departure of N2 from the "close complexes" formed by iron porphyrin and tosyl azide is nearly barrierless.For iron porphyrin nitrene,both CASSCF and unrestricted DFT calculations revealed that the triplet and open-shell singlet complexes correspond to very similar energies,and the triplet nitrene complex can be described as[(por)(-OCH3)FeII=NTs]-(?)[(por)(-OCH3)Fe?=N·-Ts]-(?)[(por)-OCH3)Fe?=N2-Ts]-.While the oss nitrene complex can be described as[(por)(-OCH3)Fe?-N·-Ts]-.In addition,the intermolecular C-H amination catalyzed by iron porphyrin nitrene follows the hydrogen atom abstraction/radical recombination mechanism with a free energy barrier of 7.1 kcal/mol on the triplet state surface.In general,the medium reactivity and easily prepared characteristic of iron porphyrin nitrene make it a potential catalyst for C-H amination.(2)Mechanistic insights into the oxidative rearrangement catalyzed by dioxygenase ChaP involved in chartreusin biosynthesisDioxygenase ChaP catalyzes the key oxidative rearrangement in the late stage of the biosynthesis of antitumor molecule chartreusin.In contrast to other common dioxygenases,for example 2,3-catechol dioxygenase,which uses the dioxygen molecule as the oxidaut,ChaP requires the flavin-activated oxygeu(O22-)as the equivalent,because the substrate(o-benzoquinone)of ChaP lacks two electrons compared with the substrate catechol.Previous experiments showed that the ChaP-catalyzed ring rearrangement contains two successive C-C bond cleavage and one lactonization,however,the detailed reaction mechanism is still unknown.In this work,the catalytic mechanism of ChaP was explored by performing quantum mechanical/molecular mechanical(QM/MM)calculations.Our calculation results reveal that ChaP uses the proximal oxygen in iron-coordinated HOO' to directly trigger the nucleophilic attack on the substrate carbonyl carbon,whereas the previous proposal that Asp49 acts as a base to deprotonate the iron-coordinated HOO-and initiates the reaction corresponds to a higher energy.In the first stage reaction,owing to the coordination with iron,the substrate is activated by accepting an electron from iron,and the resulted oxy-radical intermediate formed by O-O cleavage can easily trigger the ring arrangement.In the final decarboxylation,the phenolic anion of substrate cooperatively extracts the proton of iron-coordinated HOO-group to facilitate the nucleophilicity of the distal oxygen.These findings may provide useful information for understanding the ChaP-catalyzed oxidative rearrangement and other flavin-depedent non-heme dioxygenases.(3)Theoretical insights into the catalytic mechanism of retro-aldolase RA95.5-8FAldolase reversibly catalyzes the addition of nucleophilic donors to electrophilic receptors to form C-C bonds.Among them,the first class of aldolase catalyzes the breaking of C-C bonds by forming Schiff base intermediates.This thesis has explored the catalytic mechanism of an evolutionary efficient retro-aldolase RA95.5-8F by QM/MM method.First the protonated Lys1083 nucleophilically attacks the carbonyl group of the substrate and transfers a proton to form an alcohol,then the alcohol hydroxyl group carries a pair of electrons to bind Tyr1051 to release a water molecule and afford a Schiff base intermediate.The deprotonated Tyrl 051 abstracts a proton on the substrate to facilitate the cleavage of C-C bond to form the enamine and aldehyde.Finally,the C-N bond breaks to produce acetone and aldehyde molecules.Residues Tyr1180,Tyr1051,Lys1083 and Asn1110 always form a catalytic tetrad through hydrogen bonds in the catalytic process,which plays an important role in stabilizing the substrate and water molecule.