Fig. 6.

Protonation of the substrate orders the Met20 sidechain in the Michaelis complex. Fifty independent MD simulations of the ecDHFR:NADPH:DHF complex, with and without protonation of the N5 nitrogen, were run for 100 ns each. Kernel density estimates of the (A) hinge distance, (B) Trp22-$\varphi$, (C) donor–acceptor distance for hydride transfer change upon protonation of the substrate. These kernel density estimates were computed for each trajectory independently and the mean and 95% CI is shown for each condition. (D) The density of water around the N5 nitrogen of DHF as a function of distance from the N5 nitrogen RDF mean and 95% CI are shown. The first 50 frames (20 ns) from one trajectory are superimposed for the (E) deprotonated and (F) protonated substrate, depicting the Met20 sidechain and all waters within 4.5 Å of the N5 nitrogen of DHF. Only the initial frame is depicted for DHF and NADPH for visual clarity.