The Effect of Electrostatic Shielding on H Tunneling in R67 Dihydrofolate Reductase

Abstract

Dihydrofolate reductase (DHFR) catalyzes the hydride (H) transfer reaction between NADPH and dihydrofolate, and produces tetrahydrofolate and NADP+. R67 DHFR is a plasmid encoded enzyme, and is considered a “primitive enzyme” due to its genomic, structural, and kinetic properties.[1, 2] Interestingly, kinetic studies of R67 DHFR show an enhancement in H-transfer rate with increasing ionic strength.[3] To evaluate the source of this rate enhancement, the temperature dependency of intrinsic kinetic isotope effects (KIEs) was measured and the nature of the H-transfer step was evaluated at low and high ionic strengths. At high ionic strength, the KIEs were less temperature dependent than at lower ionic strength. These findings were evaluated using a Marcus-like model, which suggests that at higher ionic strength, the donor and acceptor of the hydride were better oriented for H-tunneling than the same system at lower ionic strength. This comparison addresses the level of system preparation that brings the reaction coordinate into a tunneling-ready conformation. While the effect is small, it is statistically significant, as apparent from the comparative data and standard deviations presented in the Supplementary Information (SI – Table S2). These data demonstrate the high sensitivity of the methodology that was developed to study this system (see detailed methods in the SI). The differences in electrostatic potential surface between low and high ionic strengths were calculated, and the theoretical findings add a molecular perspective to the experimental data.