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. Author manuscript; available in PMC: 2017 Jun 28.
Published in final edited form as: Methods Enzymol. 2016 Jun 28;577:287–318. doi: 10.1016/bs.mie.2016.05.017

Figure 8.

Figure 8

Roles of active site and distal residues on the DHFR-catalyzed reaction. Left panel: Structure of WT-ecDHFR (PDB Code 1RX2), with folate in magenta and NADP+ in light blue. A yellow arrow marks the hydride’s path from C4 of the nicotinamide to C6 of the folate, and the residues studied in ref (Singh et al., 2014) are marked as purple spheres. Right panel: Presentation of the temperature-dependence parameters of KIEint for WT (black), distal (red), and active site I14 (green) mutants of DHFR, where error bars represent standard deviation. For each point, the ordinate is the isotope effect on activation energy (ΔEa) and the abscissa is the isotope effect on the Arrhenius pre-exponential factor (AH/AT), both of which were determined from a non-linear regression of KIEint to the Arrhenius equation. The yellow block represents the semi-classical range of the Arrhenius pre-exponential factor (0.3-1.7) (Kohen, 2006). Reprinted from ref. (Singh, Francis, et al., 2015) with permission from the American Chemical Society.