Evolutionary Effects on Bound Substrate p K a in Dihydrofolate Reductase

• Published in: Journal of the American Chemical Society 2018-12, Vol.140 (48), p.16650-16660
• Main Authors: Mhashal, Anil R, Pshetitsky, Yaron, Cheatum, Christopher M, Kohen, Amnon, Major, Dan Thomas
• Format: Article
• Language: English
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.8b09089

In the present study, we address the effect of active site structure and dynamics of different dihydrofolate reductase (DHFR) isoforms on the p K 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 p K of the N5 position of the bound substrate. We observe a gradual increase in N5 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 p K 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.