Studies Of The Catalytic Mechanisms Of Methyltransferase, Peptidylarginine Deiminase And Acetolactate Decarboxylase

As we all known,enzymes are the best catalysts in natural systems.Compared with general catalysts,it has highly efficient catalysis at the physiological temperature and pressure.Furthermore,it is environment friendly.Enzymes play important role in all life activities.Therefore,understanding the mechanism of enzymes can further expand the applications of enzymes in chemical industry and our daily life.However,the enzymatic reaction is too fast to capture the details of reaction mechanism by experimental means.Recently,the quantum mechanics and molecular mechanics methods are used widely for exploring the enzymatic reactions on the basis of the developments of computer technology and theoretical chemistry methods.In this thesis,we explored catalytic mechanisms of three enzymes by employing the combined quantum mechanics/molecular mechanics(QM/MM)method.Ourcalculations revealed the catalytic mechanism,the structures of species involved in the reaction,and the energy information.Calculations will explain the experimental results,and further supplement the experiment.Moreover,the calculated results may provide theoretical information for the application of enzymes.The main research work of this thesis are as follows:(1)The catalytic mechanism of SAM methyltransferase from Escherichia coliMethyltransferase(RlmN)is a radical S-adenosylmethionine(SAM)enzyme.It is a two dual-specificity enzyme,which catalyzes the methylation of C2 of adenosine 2503 in 23S rRNA and adenosine 37 in several Escherichia coli transfer RNAs(tRNA).In this study,based on the crystal structure of RlmN in complex with tRNA substrate(PDB code:5HR6),we performed the QM/MM calculation for the catalytic mechanism of RlmN.Calculations reveal that the catalytic mechanism of RlmN includes four elementary steps.The 5'-dA radical firstly abstracts a hydrogen atom from the methylated C355 to form the C' radical,which then attacks C2 of A37,generating the paramagnetic protein-nucleic acid cross-linked intermediate(IM2).The following step is the resolution of intermediate IM2.The cleavage of C'-S' bond of C355 is concerted with the proton abstraction by C118.This step is rate-limiting with an energy barrier of 17.4 kcal/mol.Finally,C118 returns the hydrogen proton to finish this reaction.Our results may provide meaningful insight into the catalytic mechanism of RlmN and homologous enzyme Cfr.(2)The catalytic mechanism of Porphyromonas gingivalis peptidylarginine deiminasePorphyromonas gingivalis peptidylarginine deiminase(PPAD)catalyzes the citrullination of C-terminal arginines.Citrullination of arginines in proteins and peptides has an established role in physiological and pathological process.Studying the citrullination mechanism of PPAD will provide useful information for its clinical application.Firstly,we explored the proton states of active site C351 and H236 residues by QM/MM calculations.Citrullination of the peptidylarginine contains two tandem stages:deamination and hydrolysis.The energy barrier of deamination reaction is 20.17 kcal/mol.In hydrolysis process,the deprotonated D238 or H236 can act as a base to activate the hydrolytic water,which correspond to similar energy barriers(?17 kcal/mol).(3)The catalytic mechanism of acetolactate decarboxylase from Brevibacillus brevisAcetolactate decarboxylase catalyzes both enantiomers of a-acetolactate to give a single product,(R)-acetoin.(S)-acetolactate is the natural substrate,whereas(R)-acetolactate is the non-natural substrate,which corresponds to a lower conversion rate.We explored the catalytic reaction of acetolactate decarboxylase,it supplements the experimental results.The conversion of(S)-acetolacte only contatins two elementary steps:the direct decarboxylation of the substrate to form an enolate intermediate;the intermediate abstract the proton of E253 residue to generate the final product.The non-natural substrate can not directly decarboxylate.It firstly rearranged to the natural substrate(S)-acetolactate by a carboxylate migration.Then it follows the reaction mechanism of(S)-acetolactate to finish the reaction.