Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase

Abstract

The enzyme DHFR (dihydrofolate reductase) catalyses hydride transfer from NADPH to, and protonation of, dihydrofolate. The physical basis of the hydride transfer step catalysed by DHFR from Escherichia coli has been studied through the measurement of the temperature dependence of the reaction rates and the kinetic isotope effects. Single turnover experiments at pH 7.0 revealed a strong dependence of the reaction rates on temperature. The observed relatively large difference in the activation energies for hydrogen and deuterium transfer led to a temperature dependence of the primary kinetic isotope effects from 3.0+/-0.2 at 5 degrees C to 2.2+/-0.2 at 40 degrees C and an inverse ratio of the pre-exponential factors of 0.108+/-0.04. These results are consistent with theoretical models for hydrogen transfer that include contributions from quantum mechanical tunnelling coupled with protein motions that actively modulate the tunnelling distance. Previous work had suggested a coupling of a remote residue,Gly121, with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121V-DHFR, in which Gly121 was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G121V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121V-DHFR catalysis. In contrast, steady-state measurements at pH 9.5, where hydride transfer is rate limiting, revealed temperature-independent kinetic isotope effects between 15 and 35 degrees C and a ratio of the pre-exponential factors above the semi-classical limit, suggesting a rigid active site configuration from which hydrogen tunnelling occurs. The mechanism by which hydrogen tunnelling in DHFR is coupled with the environment appears therefore to be sensitive to pH.

Figure 1. Structure of DHFR from E. coli bound to NADP⁺ and H₂F

The PDB file 1RA2 [21] was used to generate the diagram. The βF-βG and the M20 loops as well as the position of the catalytically important loop residue Gly¹²¹ are indicated.

Figure 2. Fluorescence energy transfer during DHFR catalysis

Measurement of the rate of H- (light grey dots) and D- (dark grey) transfer catalysed by DHFR in a single turnover experiment measured by stopped-flow fluorescence resonance energy transfer from DHFR to the reduced cofactor at 25 °C. The relative intensity of fluorescence above 400 nm was measured after excitation at 292 nm. The fits to a double-exponential model for decreasing fluorescence intensity are also indicated. DHFR was preincubated with NADPH or NADPD and the reaction started through the addition of H₂F. Final concentrations were: DHFR, 40 μM; reduced cofactor, 20 μM and H₂F, 200 μM. The dead time of the experiments was approx. 0.002 s.

Figure 3. Arrhenius plots for H- and D-transfer during DHFR and G121V-DHFR catalysis at pH 7.0

Temperature dependence of the H- (◇) and D-transfer (●) rate constants for the reactions catalysed by DHFR (A) and G121V-DHFR (B) is shown. Each data point is the average of at least six measurements; the error bars are obscured by the symbols. Fitting the average rate value for every temperature to the Arrhenius equation yields the following parameters: DHFR, E_A^H=29.9±0.6 kJ·mol⁻¹, A_H=3.3±0.8×10⁷ s⁻¹ and E_A^D=37.7±0.6 kJ·mol⁻¹, A_D=3.07±0.80×10⁸ s⁻¹; G121V-DHFR, E_A^H=23.3±0.3 kJ·mol⁻¹, A_H=2.2±0.2×10³ s⁻¹ and E_A^D=39.0±0.6 kJ·mol⁻¹, A_D=8.1±2.0×10⁵ s⁻¹.

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.