The Role of Active Site Hydrophobic Interactions in Facilitating Catalysis in Human Terminal Deoxynucleotidyl Transferase

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase that catalyzes template-independent nucleotide addition at the 3'-end of DNA, playing a critical role in generating immune receptor diversity. While the structural importance of Loop1 in blocking template strand binding and enabling this activity is established, the precise molecular contribution of hydrophobic interactions within Loop1 to the catalytic mechanism of human TdT remains unclear. In the present study, we aim to elucidate the roles of hydrophobic Loop1 residues (L397, F400, F404) in the structural organization and catalytic function of TdT. We engineered alanine and tryptophan substitutions at these positions and systematically analyzed the resulting mutant forms using molecular dynamics simulations and pre-steady-state kinetic measurements. Our results show that substitutions L397A and F400A increase Loop1 flexibility and significantly reduce catalytic activity, particularly for purine nucleotide incorporation, while F404A completely abolishes enzymatic function. The F404W mutant largely preserves activity. All mutant forms retain the ability to bind single-stranded DNA and dNTP, but in some cases, their affinity and thermal stability were reduced. These findings demonstrate that hydrophobic interactions in Loop1 are essential for maintaining the catalytically competent conformation of TdT, ensuring precise substrate positioning and active site stability.