Role Of Substrate-Product Interactions On The Functional Dynamics Of The Adenylate Kinase

Enzyme which is mainly consist of proteins is one of the most fundamental biomolecule machines that catalyze different biological reactions.Under the action of enzyme,reactions that occur slowly could become much faster and thus play a key role in dynamic process and function.The main factor representing the catalysis ability is catalytic efficiency which depends on both the relevant chemical reaction steps and the conformation change and substrates disassociation process.All natural enzymes show a high efficiency.As a result,it has become a core problem in the biophysical field to understand the role of the enzyme in integrating the different steps of the whole cycle,overcoming the rate-limiting steps and thus achieving the high efficiency catalysis.Herein,we use a model enzyme adenylate kinase(ADK)to study the role of interaction between substrate-products in overcoming the rate-limiting step(product releasing)in the whole cycle and then explore the physical mechanism for the high efficient catalysis.In this article,by using molecular dynamic simulation,we discussed two questions,which are show below:Firstly:regulation of conformation motion of ADK by direct interaction between substrates and productsIn the catalytic cycle of ADK,the main steps usually take place in the closed state while the substrates binding and products releasing process require the catalytic sites to be exposed to the solvent as much as possible.Thus,the catalytic cycle of ADK involves the frequent switching of protein conformation change between open state and closed state.In this thesis,we used all-atom MD simulation in conjunction with H-REMD-US method to make a full study of the free energy landscape in Apo,DM,DD and TD state.Among these states,the ADP binding in the NMP domain and ATP binding in the LID domain involve direct interactions.The results show that the free energy landscape of conformation change depends on the binding state of substrates/products.In the apo state,the protein tends to be open while become closed state upon binding two products.Especially,compared to DD state,the protein tends to be open in TD state.The results above reveal that the interaction between substrate-product can facilitate the conformation opening,substrates releasing and thus accelerating the enzymatic catalysis process.Further studies show that the full opening of LID domain will change the structural intergrity of Hinge domain while the full opening of NMP domain has little impact on the Hinge domain,which implies that it's difficult to fully open LID domain in the real cycle.These results are consistent with the previous coarse-grained simulations and single molecule enzymology and can offer microscopic interaction mechanism.The results are of great significance for understanding the coupling mechanism of substrate/product binding and conformational kinetics in ADK catalytic cycle.Secondly:regulation of substrate releasing of ADK by direct interaction between substrates and productsIn the catalytic cycle of ADK,the releasing step of products in NMP domain is the rate-limiting step.How to realize the fast release of ADP in natural ADK and realize the high efficiency catalysis is an important issue.Based on the previous research results of our research group,this thesis attempts to discuss the role of substrate-product direct interaction in the release of ADP.We use three methods containg metadynamic,PacsMD and H-REMD-US to simulate the releasing process of product ADP in NMP domain and energy landscape in TD state.Our result shows that the ADP in NMP domain in TD state is easier to release and the free energy barrier is lower thanthat in DD state.And the result illustrates that the interaction of substrates and products can effectively promote the release of ADP which is the rate-limiting step during the whole cycle,which is consistent with the free energy discussed above.We further reveal that the breaking of the coordination to Mg2+ plays a key role in the release of ADP releasing.These results are of certain significance for understanding the microscopic mechanism of high catalytic efficiency of enzyme molecules in nature.The organization of the thesis is listed as below:Chapter 1 is the introduction of the background of our research topics and the molecular techniques we used in our work.In chapter 2,we study the conformation change energy landscape in Apo,DM,DD and TD state and the key roles contributing to the change in the same protein with different substrates like RMSD,SASA and contact.In chapter 3,we study the substrate disassociation in TD,DD state as well as the key factors of substrates releasing such as contact,conformation change and coordination of Mg2+.Chapter 4 is a summary of the thesis and the prospective for future studies.