Theoretical Studies On The Catalytic Mechanisms Of Several Important Enzymes

Enzymes are kinds of proteins that can act as catalysts and are produced byliving cells. Becaues of the development of the computer technology, computationalmethods have been widely used in scientific reseach fields of the enzymes. In thesisstudy, we constructed several high quality model structures of human enzymes byusing various computational techniques, such as homology modeling and moleculardynamics (MD) simulations. And the molecular binding modes has been adopted todetermine biomolecular complexes and to compare the inhibitors. We reveal themechanism of interaction between the protein and inhibitors. The finding might be agood starting point for further determination of the biological role and structure-basedinhibitor design of these human proteins.1. Computational modeling of novel inhibitors targeting the human GSTP1variants homology domainThe glutathione transferases (GSTs, E.C.2.5.1.18), also known as a family ofenzymes involved in the mechanism of cellular detoxification, catalyze thenucleophilic attack of glutathione on the electrophilic center of a number of toxiccompounds and xenobiotics. The Pi-class GST enzyme (GSTP1) has beenextensively studied because of its potential role in disease research. Previous studiespredicted four human Pi gene variants from human normal cells and malignantgliomas.We constructed a high quality model of human GSTP1*B (GlutathioneS-Transferases, Pi,1B) and revealed the interactions between protein and threeinhibitors including EA and its conjugate of Glutathione (EAG(I) and EAG(O)) toexplore the structure-function relationship by molecular dynamics (MD) simulations. Based on the results of interaction energy caculation and the analysis of MDsimulation trojactory, we identified several critical residues of stablizing the structureof G-and H-site, including Phe8, Arg13, Trp38and Tyr108. Our results also show thatthe conjutation of GSTP1*B protein may increase the binding ability of the inhibitorand the specific selectivity of Phe8and Tyr108to the substrate.Meanwhile, a detailed understanding of human GSTP1*D requires an accuratestructure, which has not been determined yet. We constructed a high quality modelstructure of human GSTP1*D by molecular dynamics (MD) simulations and revealedthe interactions between the proteins and five inhibitors including CBL, EA, EAG andLZ6to explore the structure-function relationship. We identified several criticalresidues, including Phe8, Arg13, Val35, Ile104, Tyr108, and Val113. Our resultsrevealed the specific selectivity of Phe8and Tyr108to the substrate. And we provieda new explanation for how does Ile104influence the substrate binding and ahypothesis about the indirect interaction between Val113and Tyr108. These resultsmay illustrate the alteration of enzymatic activity in the variants of GSTP1. Inaddition, the influence of Glutathione conjugate on ligands was observed.. This workwill be a good starting point for further determination of the biological role andstructure-based inhibitor design of human Pi-class GST.2. A Molecular Dynamics and Computational Study of Human KAT3homologymodel Involved in KYN Pathway and substrate binding studyKynurenine aminotransferase III (KAT3) is a novel member of the kynurenineaminotransferase enzyme family. Its active site topology and structure characteristicshave not been established. In this study, with extensive computational simulations,including homology modeling and molecular dynamics simulations, a3D structuremodel of human KAT3(HKAT3) dimmer was created and refined. Furthermore,CDOCKER approach was employed to dock two ligands (L-methionine andL-tryptophan) into the active sites of human KAT III dimmer and uncover theligand-binding modes. The complexes were subjected to5ns MD simulation, and the results indicate that Tyr159and Trp53might be the key residues as they have the largecontributions to the binding affinity, which is in good agreement with theexperimental results. Moreover, another two residues (Asp160and Tyr97) are alsofound that their strong interactions stabilize the whole system. The structural andbiochemical insights obtained from the present study will be helpful for designing thespecific inhibitors of HKAT3.However, KATs also catalyze the transamination of kynurenine (KYN) pathwayand endogenous KYNs have been suggested to correlate highly to abnormal braindiseases. HKAT3is a key member of KAT family, while the binding mechanism ofKYN and cofactor with HKAT3has not been determined yet. In this study, we focuson the structure-function relationship among KYN, cofactor and HKAT3. The bindingmodels of KYN complex and KYN&cofactor complex were obtained and werestudied by Molecular Dynamics (MD) simulations. We identified several criticalresidues and influence of conformational changes in human Kynurenineaminotransferase3complexes. The cofactor may play significant contributions notonly to the catalysis, but also to the binding. In addition, a hypothesis is proposed thata strong hydrophobic interaction between Tyr159and Lys280may influence thebinding mode and the binding region of the substrate and the cofactor. Our results willbe a good starting point for further determination of the biological role.3. Building KCNQ1/KCNE1docking models and probing their interactions bymolecular dynamics simulationsThe slow delayed-rectifier (IKs) channel is composed of KCNQ1(pore-forming)and KCNE1(auxiliary) subunits, and functions as repolarization reserve‘in the heart.Design of IKs-targeting anti-arrhythmic drugs requires detailed3-D structures of theKCNQ1/KCNE1complex, a task made possible by Kv-channel crystal structures(templates for KCNQ1homology modeling) and KCNE1NMR structures. PreviousKCNQ1/KCNE1models included only the KCNE1transmembrane domain(E1-TMD), despite data indicating the importance of extracellular N-terminal domain (E1-NT) in IKs function. Our goal is to build KCNQ1/KCNE1models includingE1-NT:(a) creating KCNQ1homology model based on new experimental restraints,(b) refining KCNE1NMR structures to correct non-native loop configurations,(c)docking E1-TMD and E1-NT to KCNQ1in a step-wise manner,(d) testing modelpredictions independent from restraints used in model-building,(e) subjecting thecomplex to molecular dynamics simulations in explicit lipid/solvent environment, andanalyzing KCNQ1/KCNE1contacts and impact of docking on their backbonefluctuations. Our analysis suggests two novel aspects of KCNQ1/KCNE1interactions:(1) Frequent contacts between E1-NT and KCNQ1S5-P linker may serve to stabilizethe docking conformation and enhance IKs expression in cell surface membrane,(2)E1-TMD uses one helical face of high backbone fluctuations to optimize contactswith KCNQ1during protein docking.