Evolutionary Effects On Bound Substrate Pk(A) In Dihydrofolate Reductase

In the present study, we address the effect of active site structure and dynamics of different dihydrofolate reductase (DHFR) isoforms on the pK(a) of the bound substrate 7,8-dihydrofolate, in an attempt to understand possible evolutionary trends. We apply a hybrid QM/MM free energy perturbation method to estimate the pK(a) of the NS position of the bound substrate. We observe a gradual increase in NS basicity as we move from primitive to more evolved DHFR isoforms. Structural analysis of these isoforms reveals a gradual sequestering of water molecules from the active site in the more evolved enzymes, thereby modulating the local dielectric environment near the substrate. Furthermore, the present study reveals a clear correlation between active site hydration and the N5 pK(a) of the substrate. We emphasize the role of the M20 loop in controlling the active site hydration level, via a preorganized active site with a more hydrophobic environment and reduced loop flexibility as evolution progresses from bacterial to the human enzyme.