Hydride Transfer in DHFR by Transition Path Sampling, Kinetic Isotope Effects, and Heavy Enzyme Studies

Abstract

Escherichia coli dihydrofolate reductase (ecDHFR) is used to study fundamental principles of enzyme catalysis. It remains controversial whether fast protein motions are coupled to the hydride transfer catalyzed by ecDHFR. Previous studies with heavy ecDHFR proteins labeled with (13)C, (15)N, and nonexchangeable (2)H reported enzyme mass-dependent hydride transfer kinetics for ecDHFR. Here, we report refined experimental and computational studies to establish that hydride transfer is independent of protein mass. Instead, we found the rate constant for substrate dissociation to be faster for heavy DHFR. Previously reported kinetic differences between light and heavy DHFRs likely arise from kinetic steps other than the chemical step. This study confirms that fast (femtosecond to picosecond) protein motions in ecDHFR are not coupled to hydride transfer and provides an integrative computational and experimental approach to resolve fast dynamics coupled to chemical steps in enzyme catalysis.

Figure 1

DHFR-catalyzed hydride transfer reaction and previous T-d KIE data. (A) Overall reaction catalyzed by DHFR. The blue dots in the structures indicate the boundary atoms that divide the substrates into QM and MM regions in our atomistic simulations. (B) Previously reported intrinsic deuterium isotope effects (^Dk_hyd) on the hydride transfer of l- and h-DHFRs (blue and red solid lines) at pH 9. The present report examines the difference between l- and h-DHFR deuterium KIEs at 5 °C.

Figure 2

Temperature and ecDHFR protein isotope effects alter subpicosecond protein dynamics (Figure S2) but not the hydride transfer TS. (A) l- and h-DHFRs show the same reactive trajectories projected on the d_DH and d_HA distances. The thick black line in each plot marks the TS location determined by committor analysis (Figure S1). (B) Superimposition of four representative TS structures (purple: light 280 K; red: light 300 K; blue: heavy 280 K; green: heavy 300 K) indicates the same TS geometry for l- and h-DHFRs. The TS structures shown in this figure are extracted from the reactive trajectories shown in Figure S1.

Figure 3

Temperature and DHFR mass effects on the pre-steady-state rate constants (k_burst; A) and deuterium KIEs (^Dk_hyd; B) measured by stopped-flow experiments at pH 7 (blue: l-DHFR; red: h-DHFR). The pre-steady-state ^Dk_hyd values (B) are within the experimental errors of intrinsic KIEs measured by competitive experiments at pH 7 (data from ref 38). The k_burst and ^Dk_hyd values are also listed in Table S2.

Scheme 1. Kinetic Mechanisms of l-DHFR (Blue) and h-DHFR (Red) at 5 °C, pH 9^a

^aUnder saturating substrate concentrations, the cofactor NADPH predominantly binds to DHFR·THF complex during the catalytic turnover instead of the apoenzyme, except during the first turnover (k₁ and k₋₁, green).