Theoretical Studies On The Catalytic Mechanisms Of Several Important Enzymes

Enzymes are catalysts that produced by living cells. Virtually all of themare protein. Enzymes belong to the biological macromolecule, but the regionthat binds the substrate and converts it into product is a relatively small part.This region is known as the active center. The enzyme combines with itssubstrate to form an enzyme-substrate complex. It is an extremely unstablestate and has the highest free energy in the reaction pathway. This transientspecie is difficult to capture by the experimental techniques. Computersimulation can make up the shortfall and give the detailed information of thecatalytic reaction on the atomic level. By analysis of the intermediates andtransition states, and binding the kinetic method to determine the rate-limitingstep, we can infer the catalytic mechanism of the enzymes.1. The mechanism of rearrangement reaction catalyzed byN5-carboxyaminoimidazole ribonucleotide mutaseIn this paper, quantum chemistry calculation methods were used. Thecluster model for the simulations was constructed on the basis of crystal structure. We have studied the catalytic mechanism ofN5-carboxyaminoimidazole ribonucleotide mutase, glutathione transferaseGtt2 andα-Methylacyl-CoA racemase using theoretical method. The mainresults are summarized as follows:De novo purine biosynthesis is central to all life forms except protozoa.In this process, the carboxylationof 5-aminoimidazole ribonucleotide (AIR) isconverted to 4-carboxyaminoimidazole ribonucleotide (CAIR) catalyzed byClass I PurE. The necessity of Class I PurE for the growth of microbe hasbeen shown by genetic studies. Deletion of this enzyme will result in a purineauxotroph that is unable to propagate in human or mouse serum and is notviable in animal models predictive of disease.In the present study, the complete reaction mechanism of PurE has beeninvestigated by using the density functional theory with the hybrid B3LYPfunctional. The model containing 93 atoms was constructed on the basis ofthe X-ray structure of PurE from Escherichia coli. Additionally, we hadpreceded the charge population analysis with the method of the Natural BondOrbital (NBO) to verify the soundness of the proposed mechanism. Theresults show that this rearrangement reaction is carried out in two steps, one isdecarboxylation and the other is carboxylation. The conserved residue His45plays an essential catalytic role as both general aicd and general base. Atetrahedral intermediate iso.CAIR is observed during the carboxylationreaction and it is a plausible construction proved in experiment. The potential energy curves are drawn in terms of single-point energies. By comparing theenergy barriers, we could consider the carboxylation step as the rate-limitingstep. Furthermore, other residues including Ser43, Ala44, His45, Gly71,His75 and Leu76 also play important roles via the strong hydrogen-bondinteractions.2. Reaction mechanism ofα-Methylacyl-CoA racemase: Atheoretical investigationThe branched-chain fatty acids are important component in the humandiet. Also, they are used for non-steroidal anti-inflammatory drugs such asibuprofen. In their metabolic process, only the (S)-substrate can bemetabolized through the branched-chain acyl-coA oxidases.α-Methylacyl-CoA racemase (AMACR) catalyzes the conversion fromR-enantiomer to S-enantiomer at the 2-position of fatty acyl-CoA esters.Therefore, AMACR is the essential enzyme in the metabolic process. Inaddition, AMACR can be used as highly sensitive and specific marker forprostate cancer. It has very important clinical value in the early diagnosis ofprostate cancer. Subsequently, the over-expression has also been found inbreast, colorectal and ovarian cancer.In this paper, amethylacyl-CoA racemase from Mycobacteriumtuberculosis (MCR) were studied. The amino acid sequence identity betweenMCR and human is up to 43%. The calculation was completed by using thedensity functional theory (DFT) B3LYP method. The geometry optimizations were done with 6-31G (d) basis set and the single-point energy calculationswere done with 6-311 + + G (2d, 2p) basis set. Our calculation resultsillustrate the catalytic mechanism of MCR, and support the experimentalresults proposed by Prasenjit Bhaumik. His126 and Asp156 function as thegeneral base and the general acid respectively in the 1, 1 - proton transferreaction. It can be seen from the optimized structure, the intermediate is aplane enolate anion type, so the type of the enol or ketene is excluded. Bycomparing the energy barrier, we consider that the conversion from theS-enantiomer to the R- enantiomer is the rate-limiting step during theracemization. The theoretical studies on the mechanism of 1, 1 - protontransfer reaction catalyzed by MCR will be helpful to synthetize theanti-cancer drugs which designed based on the transition state structure.3．The theoretical study on the GSH activation mechanismGlutathione S-transferase (GSTs) can cause the metabolism ofelectrophilic toxic substances. So it has an important role in detoxification.Glutathione (GSH) can react with a range of electrophilic toxic substances,and generate the non-toxic soluble substances, then the products are excludedfrom the cell. However, the drug metabolism will result in drug resistance,even the failure of clinical treatment. Before it reacting with the electrophilicsubstrates, GSH should be activated into the thiolate form. Recently, the GSHactivation mechanism catalyzed by typical GSTs (GSTA1-1, GSTP1-1 andGSTM1-1) has been proposed. How about the GSH activation mechanism catalyzed by the atypical GST (Gtt2)?In the present study, all geometry optimizations, single-point calculationsand frequency analysis were performed using density functional theory (DFT)with the B3LYP functional and the basis set 6-31G (d) as implemented inGaussian03. The GSH activation mechanism of Gtt2 has been investigatedand the results are consistent with the experimental data available. The protontransfer mediated by a water molecule is energetically feasible. Theimportance of water molecule as promoter is unequivocal. His133 functionsas general base in the reaction. The reason why the product has a higherenergy has been analyzed by comparing with the typical GSTs. In addition,another model that including a smaller amount of water molecules wasselected. The effects of water molecules in the system were investigated. Itproved that different model does not cause the changes of the pathway, but theenergy and configuration will be affected.