Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrumpernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 a...Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrumpernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 and formed hydrogen bond with Ile567. To study the effect of Tyr444 on the activity of APE1547, site-directed mutagenesis was applied. Two mutant enzymes T444S and T444G were created. Comparison of the mutant enzymes with wide enzyme, the thermostability of mutants T444S and T444G decreased by 10%-20%, but the catalytic efficiency of mutants toward pNPC8 and Ac-Leu-pNA increased 1.33 and 1.75 fold respectively. Molecular modeling shows that the elimination of hydrogen bond between Tyr444 and Ile567 is the cause of the decrease in thermostability and increase in catalytic efficiency. These observations suggest that Tyr444 plays an important role in the catalytic ability and thermostability of this enzyme.展开更多
基金Supported by the National Natural Science Foundation of China(Nos.30400081 and 20432010)
文摘Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrumpernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 and formed hydrogen bond with Ile567. To study the effect of Tyr444 on the activity of APE1547, site-directed mutagenesis was applied. Two mutant enzymes T444S and T444G were created. Comparison of the mutant enzymes with wide enzyme, the thermostability of mutants T444S and T444G decreased by 10%-20%, but the catalytic efficiency of mutants toward pNPC8 and Ac-Leu-pNA increased 1.33 and 1.75 fold respectively. Molecular modeling shows that the elimination of hydrogen bond between Tyr444 and Ile567 is the cause of the decrease in thermostability and increase in catalytic efficiency. These observations suggest that Tyr444 plays an important role in the catalytic ability and thermostability of this enzyme.
