Study on a Specific Site Tyr(444) on a Hyperthermophilic Enzyme APE1547

Hyperthermophilic enzyme APE 1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrum pernix K1. The Tyr(444) located in the catalytic domain adjacent to the catalytic amino acid Ser(445) and formed hydrogen bond with Ile(567). To study the effect of Tyr(444) on the activity of APE 1547, 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 Tyr(444) and Ile(567) is the cause of the decrease in thermostability and increase in catalytic efficiency. These observations suggest that Tyr(444) plays an important role in the catalytic ability and thermostability of this enzyme.