Sandbox Reserved 1846: Difference between revisions

Ser 283 and Asp 238, are located outside of the binding pocket in the <scene name='10/1075247/Start_material_for_s283_and_d2/2'>Reference Ser283 and Asp238</scene>.Two wild-type residues, <scene name='10/1075247/S283-d238/5'>Ser283 and Asp238</scene>, were engineered to form a disulfide bond by replacing them with Cys. This decision was based on their spatial proximity in the 3D structure and their location in a region that resembles metal-binding sites in homologous PET-degrading enzymes. Unlike those metal-dependent sites, the LCC structure lacked coordinated ions. For that reason, the researchers engineered a covalent linkage instead to increase thermal stability without requiring additives like calcium. The wild-type protein has a melting point of 84.7°C, while the <scene name='10/1075248/C283-c238/2'>S283C and D238C mutant</scene> increased the melting point to 94.5°C, a 9.8°C improvement, which is higher than any other mutations. However, this increase in stability was accompanied by a 28% decrease in enzymatic activity compared to the wild-type. This trade-off between stability and activity shows the balance in enzyme engineering, as increasing structural integrity can sometimes restrict the flexibility needed for catalytic function.