After the initial setup of the complexes in WT and its mutants, systems were minimized in two steps for removing the bad contacts. In the step 1, only solvent molecules were minimized using 5000 steps of steepest descent followed by a 5000 steps of conjugate gradient minimizer. In the step 2, all the complexes were minimized without any constraints using the same minimizer as in step 1. All systems were then gently annealed from 10 to 300 K under NVT ensemble for 50 ps with a weak restraint of 5 kcal/mol/Å 2 . Subsequently a density equilibrium was performed using NPT ensemble at targeted temperature of 300K and pressure 1 atm for 1ns using the Langevinthermostat and Berendsen-barostat, with a weak restraint of 1 kcal/mol/Å 2 . Thereafter, we removed all the restrains and the systems were further equilibrated for 3 ns followed by 50 ns of production simulations for each system with TSA and 100 ns for Michaelis Complex. The covalent bonds that contain hydrogens were constrained using SHAKE algorithm, and a particle mesh Ewald (PME) was utilized to treat long-range electrostatic interactions during all the MD simulations . An integration step of 2 fs was used during the entire simulation. All the MD simulations were performed with the GPU version of the Amber 20 package .