Solvent Effects on Environmentally Coupled Hydrogen Tunnelling During Catalysis by Dihydrofolate Reductase from Thermotoga maritima

Abstract

Protein motions may be perturbed by altering the properties of the reaction medium. Here we show that dielectric constant, but not viscosity, affects the rate of the hydride-transfer reaction catalysed by dihydrofolate reductase from Thermotoga maritima (TmDHFR), in which quantum-mechanical tunnelling has previously been shown to be driven by protein motions. Neither dielectric constant nor viscosity directly alters the kinetic isotope effect of the reaction or the mechanism of coupling of protein motions to tunnelling. Glycerol and sucrose cause a significant increase in the rate of hydride transfer, but lead to a reduction in the magnitude of the kinetic isotope effect as well as an extension of the temperature range over which "passive" protein dynamics (rather than "active" gating motions) dominate the reaction. Our results are in agreement with the proposal that non-equilibrium dynamical processes (promoting motions) drive the hydride-transfer reaction in TmDHFR.

Update 1 of: Tunneling and Dynamics in Enzymatic Hydride Transfer

“…Of particular interest, it has been demonstrated that increased solvent viscosity further increases the temperature dependence of the kinetic isotope effect at reduced temperatures for tm-DHFR. 135 Comparable viscosity effects have not been detected previously for other enzyme systems, although a surface-modified variant of glucose oxidase (section 4) shows reduced values of A H / A D relative to wild-type. The sensitivity to viscosity seen for tm-DHFR may be a consequence of finely tuned intersubunit interactions, which are compromised by a combination of nonphysiological temperature and solvent conditions.…”

Probing coupled motions in enzymatic hydrogen tunnelling reactions

“…We recently embarked on a study to test this proposal with TmDHFR, using a range of organic co-solvents [41]. Our study revealed no directly proportional effect from the solvent viscosity on either the steady-state rate (k cat ) or the rate of hydride transfer (k H ).…”

“…A separate line of best fit is shown for the rate constants of hydride transfer, k H , as a function of glycerol concentration. Data taken from [41].…”

Thermal Adaptation of Dihydrofolate Reductase from the Moderate Thermophile Geobacillus stearothermophilus

“…The pH was adjusted after the addition of salts and cosolvents. Details of dielectric constants and viscosities of solvent mixtures are from ref (32). …”