In the "well-tempered" variant (WTMD), each Gaussian is scaled down over time by a bias factor 𝛾 = 𝑒 " $(&,() * + ∆-, which is defined by the value of 𝑤(𝑠, 𝑡) at the same point where the Gaussian is centered, the Boltzmann constant 𝑘 0 , and a biasing temperature ∆𝑇 selected as an input parameter. This procedure determines the deposition of Gaussians with different heights, which depend on the biases already accumulated in the given point of the CV-space. Here, WTMD was implemented with the PLUMED 2.9 plug-in. Gaussians were deposited with a rate 𝜔 = 50, setting the Gaussian height at 5 kJ/mol, 𝛿𝑠 at 0.05 Å and 𝛾=20. WTMD QM/MM MD simulations were performed with the sander engine implemented in AMBER adopting the same theory level (DFTB3) and setup used for unbiased QM/MM MD simulations. Each reaction step (acylation and deacylation for PETase-PET4 and PETase-PEF4 systems) was simulated for 1000 ps, followed by reweighting of the biased distribution. The reweighting and block analysis were used to monitor the convergence and to calculate the errors in the FE estimation.