Theoretical Insights Into Catalytic Mechanisms Of Two Iron-containing Enzymes

Enzymes are the basis of all life activities and the stimulating promoter of all chemical changes.Proteases consist mainly of 20 amino acids.Enzymes are excellent catalysts and they can increase the reaction rate in the range of 5-17 orders.Catalytic mechanism of enzymatic reaction includes acid-base catalysis,metal ion catalysis,covalent catalysis and so on.Enzyme reaction systems are complex and most of the reactions are rapid.It is difficult to determine the structures of intermediates and transition states only by experimental means.Theoretical chemistry has unique advantages in the enzymatic reaction.The calculated results can complement the experimental work and play an indispensable role in understanding the enzymatic reaction.In this paper,molecular dynamics simulation and QM/MM method were used to study the catalytic reaction of two enzymes.On the basis of the crystal structure,a model has been constructed.The possible pathway of reaction has been determined by calculations.The structural characteristics and energetic information of intermediates and transition states involved in the reaction have been analyzed.These results establish a theoretical foundation for the further study of enzyme-catalyzed reactions.The main research contents of this dissertation as follows:(1)Insights into the catalytic mechanism of NOV1NOV1 is a stilbene cleavage oxygenase(SCO)responsible for the oxidative cleavage of the central double bond of stilbenes,forming two phenolic aldehydes.In the active site of NOVI,the iron cofactor is coordinated by four histidines and no negatively charged ligand,which is unusual for iron-containing enzymes.Here,on the basis of the recently obtained crystal structure of NOV1,we performed quantum mechanics/molecular mechanics(QM/MM)calculations to elucidate the reaction mechanism of NOV1.According to our calculations,two binding modes of 02 to Fe(?)(end-on and side-on modes)have been recognized,and the end on mode is suitable for catalysis.The side-on mode,which has been obtained from the crystal,is an inactive form,which should firstly change to the end mode to initiate the catalytic reaction.For the reactant in end on mode,the triplet and quintet states correspond to very similar energies.Of the three considered pathways,two pathways(path a and path_b1,which differ in the order of formation of first C-O bond)undergoing the dioxetane intermediate are calculated to be favorable,which can confirm the dioxygenase mechanism of NOV1.However,the pathway(path b2)involving the epoxide intermediate corresponds to high energy barriers for the formation of second C-O bond(27.49 and 31.56 kcal mol-1 on the triplet and quintet state surfaces),and can be ruled out.However,in path_b2,the formation of the epoxide intermediate is easy,which may react with the solvent water(non-enzymatic reaction)to form the byproduct.In addition,the different protonation states of 4'-OH of substrate and K134,as well as the coordination of additional water molecule with the iron do not significantly influence the reaction barrier.(2)The mechanism of desaturation,ring rearrangement and hydroxylation by the PrhA(V150L/A232S)/AusEPrhA is a nonheme Fe?a-ketoglutarate-dependent oxygenase from Penicillium brasilianum.It can catalyze many kinds of oxidation reactions.General catalytic mechanism of PrhA(V150L/A232S)/AusE has been proposed,but the details of the mechanism are still unknown.Here,on the basis of the crystal structure of PrhA(V150L/A232S),the reactant model was constructed.We performed quantum mechanics/molecular mechanics(QM/MM)calculations to clarify the catalysis mechanism of PrhA(V150L/A232S)/AusE.According to the calculations,the whole catalytic reaction can be divided into three continuous reactions:desaturation,carbon skeleton rearrangement to spiro-lactone system and hydroxylation.These reactions are initiated by abstracting H atoms.In the quintet,the energy barrier of abstracting H1 atom is 19.4 kcal mol-1.In the rearrangement reaction,the energy barrier of ing H3 atom from the C3 is 23.6 kcal mol-1.The energy barrier of abstracting H6 atom is 21.8 kcal mol-1 in the hydroxylation.We speculate that the main reason for the different barriers is the different relative positions(distances or angles)of oxygen atoms and hydrogen atoms in Fe?=O or Fe?-OH intermediates.The functional convertion between PrhA and AusE may be caused by the different positions between Fe?=O complex and the H atoms of substrate.