Figure 4. Temperature dependence of the KIEs for H- and D-transfer from NADPH/NADPD to H₂F.

(A) Results from pre-steady-state measurements at pH 7.0 for catalysis by DHFR (●) and G121V-DHFR (◇). The best fit through the data yielded ΔE_A^H/D of 7.83±0.98 kJ·mol⁻¹ and A_H/A_D of 0.109±0.043 for DHFR and ΔE_A^H/D of 15.83±0.44 kJ·mol⁻¹ and A_H/A_D of 0.0024±0.0004 for G121V-DHFR. (B) Steady-state measurements for DHFR catalysis at pH 9.0 and 5.0. The best fit through the data between 15 and 35 °C is indicated and yields ΔE_A^H/D of 1.11±0.79 kJ·mol⁻¹ and A_H/A_D of 1.81±0.19.