Abstract
A key question concerning the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) is whether the Met(20) loop is dynamically coupled to the chemical step during catalysis. A more basic, yet unanswered question is whether the Met(20) loop adopts a closed conformation during the chemical hydride transfer step. To examine the most likely conformation of the Met(20) loop during the chemical step, we studied the hydride transfer in wild type (WT) ecDHFR using hybrid quantum mechanics-molecular mechanics free energy simulations with the Met(20) loop in a closed and disordered conformation. Additionally, we investigated three mutant forms (I14X; X = Val, Ala, Gly) of the enzyme that have increased active site flexibility and donor-acceptor distance dynamics in closed and disordered Met(20) loop states. We found that the conformation of the Met(20) loop has a dramatic effect on the ordering of active site hydration, although the Met(20) loop conformation only has a moderate effect on the hydride transfer rate and donor-acceptor distance dynamics. Finally, we evaluated the pK(a) of the substrate N5 position in closed and disordered Met(20) loop states and found a strong correlation between N5 basicity and the conformation of the Met(20) loop.