| Extremophilic enzymes are enzymes capable of performing catalytic function at extremely high or low temperatures.Extremophilic enzymes often have different thermodynamic activation parameters with respect to those of their mesophilic counterparts.However,the structural origin of the the specific thermodynamic activation parameters for the extremophilic enzymes still remains unclear.The aims of this thesis are as follows:?)to establish a calculation pipeline for obtaining the thermodynamic activation parameters(activation free energy,activation enthalpy and activation entropy)of the enzymatic catalytic reaction based on the existing methods and theories so as to lay a methodological basis for future study on the adaptation mechanisms of extremophilic enzymes in laboratory;?)to explore the structural factors causing the specific thermodynamic activation parameters of extremophilic enzymes and to identify the key residues that contribute substantially to the reduction of activation free energy;?)to clarify the specific way by which the psychrophilic enzyme lowers its activation free energy.The study objects of this study are two differently temperature adapted orthologous members from the trypsin-like serine protease family,i.e.,the cold-adapted AST(anionic salmon trypsin)and the warm-active BT(bovine trypsin),respectively.The activation free energies(?G(?))for catalytic reactions of these two enzymes at 300 K were obtained using the empirical valence bond(EVB)calculation combined with umbrella sampling(US)simulations.Furthermore,the activation enthalpies and activation entropies for the catalytic reactions of these two enzymes at 300 K were obtained through linear-regression analysis of the high-precision Arrhenius plots,which were obtained using EVB/US simulations at different temperatures.Our results show that,at 300 K,the cold-adapted AST has a slightly lower activation free energy(?G(?)=18.2 ± 1.6 kcal/mol),a significantly lower activation enthalpy(?H(?)= 13.6±3.4 kcal/mol),and a more negative activation entropy(T?S(?)=-4.6 ±3.4 kcal/mol)than the respective corresponding parameters(?G(?)=19.0±1.4,?H(?)=27.1 ± 4.4,TAS(?)= 8.1 ±4.4 kcal/mol)of BT,and this is in agreement with the expectations from the transition state theory.Since temperature lowering will lead to an exponential decrease in the catalytic reaction rate of a enzyme,the cold-adapted enzyme will reduce their activation free energy for purpose of maintaining a high catalytic rate at low temperature;as a matter of fact,the reduction of activation free energy is mainly achieved by reducing the activation enthalpy,and the relation of ?G = ?H-T?S determines that the lowering of activation enthalpy will inevitably lead to a lower(or more negative)activation entropy.-It is generally believed it is the higher conformational flexibility of the cold-adapted enzyme compared to that of its mesophilic/thermophilic counterparts that maintains the high catalytic activity of the psychrophilic enzyme at low temperatures,and the reason for this is that the high flexibility reduces the activation enthalpy of psychrophilic enzymes.However,the questions of whether the changes in global flexibility,local flexibility,or the flexibility of individual amino acid residues will exert a larger influence on the catalytic activity still need to be analyzed in detail.In order to study the relationship between flexibility of the side-chain/main-chain of a single residue and the enzymatic catalytic activity,the most flexible residue Tyr97 in the psychrophilic enzyme was selected for restraining atomic fluctuations of its main chain and side chain,respectively,and the thermodynamic activation parameters of the psychrophilic enzyme were then calculated under these two conditions,respectively.The results demonstrate that the three activation parameters for AST with the restrained main-chain fluctuations in Tyr97 remain almost unchanged when compared to those of the AST without any fluctuation restriction,while when the side-chain fluctuations of Tyr97 was restrained,collectively obvious increases in all three parameter of AST were observed,showing a trend towards the activation parameters of the mesophilic BT.This indicates that the maintenance of the side-chain flexibility/mobility of a single crucial residue plays an important role in maintaining the catalytic activity of the cold-adapted AST.Since the activation free energy is the difference between the free energies of reactant state and the transition state,there seems to exist three ways by which an enzyme could reduce its activation free energy:?)the free energy of the reactant state remains unchanged,while the free energy of the transition state decreases(transition-state stabilization);?)the free energy of the reactant state increases,while the free energy of the transition state remains unchanged(reactant-state destabilization);?)the free energy of the reactant state increases,while the free energy of the transition state decreases(both i)and ?)work simultaneously).In order to clarify the specific way by which the cold-adapted AST lowers its activation free energy,we calculated the"solvation" free energy(which is the electrostatic free energy contribution of the surrounding protein and water to the reacting fragment)values for transition state and reactant state of both the psychrophilic and mesophilic trypsins and consider the"solvation" free energy as an approximation of the absolute free energy value.The results show that the reactant states of the psychrophilic and mesophilic enzymes have similar free energy values,while the transition state of the psychrophilic enzyme has a lower value,thus indicating that AST reduces its activation free energy through transition-state stabilization.Finally,the electrostatic activation free energy(i.e.,the electrostatic contribution of a residue to the activation free energy)values of each residue in these two proteases were calculated using the local reaction field(LRF)method to attempt to identify residues that likely make a significant contribution to the reduction of the activation free energy.The results show that Glyl93 has a more negative electrostatic activation free energy value in both BT and AST,indicating that this residue makes a significant positive contribution to the lowering of activation free energy;Ser214 has the highest electrostatic activation free energy values in both enzymes,indicating that it makes a substantial negative contribution to the reduction of the activation free energy.In summary,we have established in this thesis a calculation pipeline for obtaining thermodynamic activation parameters of enzymatic catalytic reaction based on the EVB/FEP/US simulation,proved that the side-chain fluctuations of the key high-flexibility residues distant from the catalytic active center play an important role in maintaining the catalytic activity of the cold-adapted AST,clarified the way by which AST reduces its activation free energy is the transition-state stabilization manner,and identified the key residues that contribute significantly to lowering of AST activation free energy.The results of this study are of great significance to further investigating and understanding the cold adaptation mechanisms/origin of psychrophilic enzymes,in particular from perspectives of thermodynamics,kinetics and energetics;in addition,the key residues identified in this thesis provide valuable information for rational modification of trypsin. |
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