| Thermostability at high temperature is an inherent property of hypertherm ophilic enzymes. Hyperthermophilic enzymes have become model systerms to study enzyme evolution, enzyme stability and activity mechanism, protein structure-function relationships, and biocatalysis under extreme conditions. In addition to their high thermostability, they show a higher resistance to chemical denaturants. In view of these important advantages, thermozymes are attracting much industrial interest for it is essential for designing more stable enzymes for industrial processes. Recently, Site-directed mutagenesis (SDM) experiments, comparisons of structure and stability of thermozymes and mesozymes as well as structure analysis have revealed some important factors that contribute to the remarkable stability of thermozymes including additional intermolecular interactions and faverable general conformational structure. However, no single universal mechanism has been found so far that promotes stability.The recombinant protein APE1547 from the thermophilic archaeon Aeropyrum pernix K1, shows extremely high thermalstability and resistance to chemical denaturants. It shows both esterase activities and acylamino acid-releasing enzymatic activities. By sequence alignment, we found that APE1547 have high homology to prolyl oligopeptidase (POP) APE1547 consists of two domains, which are the N-terminalβ-propeller domain and C-terminalα/βhydrolase domain. Theβ-propeller domain has high stability and low sequence homology between distinct enzymes.To determine the contribution of hydrogen-bond inβ-propeller domain to the stability of APE1547, we creat mutants T127V, R145V and T127V/R145V, which are depleted of three hydrogen-bondings, Thr127-Gly154, Leu182-Arg145 localed in the blades 3 and 4 within the propeller domain. The optimum temperature of wild type is 95℃or more, while that of mutants T127V, R145V are decreased to 92.5℃.The optimum temperature of double mutants is even decreased to 90℃.There is no difference between wide type and mutants in optimum pH that is 8.0, except that double mutants performed in a wider range compared to wide type. Their substrate specificities are consistent. Kinetic analysis revealed that the catalytic efficiency kcat/Km values for T127V, R145V and T127V/R145V are 107.5%, 117.3% and 127.5% of that of the wide type, respectively, which suggests that the catalytic abilities for the mutants are improved.The difference in structure between the mutants and wide type is detemined by CD and Intrinsic fluorescence, hydrophobic fluorescence probe. The simultaneous removal of three hydrogen bonds destoried some of theβ-strands and made the tertiary structure significantly flexible, whereas T127V and R145V have little effects on the structure. In order to study the stability of APE1547, we determined the kinetic stability and thermodynamic stability. The half-life of denaturation of wide type at 85℃is 15.26 h. In the case of T127V, R145V and T127V/R145V, they are 11.33, 10.37 and 4.9 h, respectively. The free energy is decreased 1.14, 0.88 and 3.55 kJ/mol respectively. The thermal stability of wide type and mutants has been characterized thermodynamically by differential scanning calorimetry(DSC). By showed that the enzyme has two thermodynamically independent domains. As recorded by DSC, the Tm of T127V, R145V and T127V/R145V are decreased 2.2, 4.4 and 9.1℃respectively. The changes in the activity and conformation of wide type and mutants were determined during unfolding by guanidine-HCl(Gdn-HCl).The result shows that the unfolding of wide type and mutant follows three-state folding patterns which means that the two domains unfold at different Gdn-HCl concentration scope .The differernce in Tm between the two domain is decreased of the mutants, the double mutants was more less.The unfouding free energy of T127V, R145V and T127V/R145V were decreased 4.35, 6.25 and 10.53 kJ/mol respectively.In this study, we removed three hydrogen bondings that localed in blades 3 and 4 within the propeller domain and proved the contribution of hydrogen bonds to the stability of enzyme molecule. This paper may enrich the stability mechanism of Hyperthermophilic enzymes and provides the foundation for designing the structure of proteins with higher stabilization.
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