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Temperature-Dependent Kinetic Isotope Effects in R67 Dihydrofolate Reductase from Path-Integral Simulations - PubMed

  • ️Fri Jan 01 2021

Temperature-Dependent Kinetic Isotope Effects in R67 Dihydrofolate Reductase from Path-Integral Simulations

Anil R Mhashal et al. J Phys Chem B. 2021.

Abstract

Calculation of temperature-dependent kinetic isotope effects (KIE) in enzymes presents a significant theoretical challenge. Additionally, it is not trivial to identify enzymes with available experimental accurate intrinsic KIEs in a range of temperatures. In the current work, we present a theoretical study of KIEs in the primitive R67 dihydrofolate reductase (DHFR) enzyme and compare with experimental work. The advantage of R67 DHFR is its significantly lower kinetic complexity compared to more evolved DHFR isoforms. We employ mass-perturbation-based path-integral simulations in conjunction with umbrella sampling and a hybrid quantum mechanics-molecular mechanics Hamiltonian. We obtain temperature-dependent KIEs in good agreement with experiments and ascribe the temperature-dependent KIEs primarily to zero-point energy effects. The active site in the primitive enzyme is found to be poorly preorganized, which allows excessive water access to the active site and results in loosely bound reacting ligands.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1

(A) Schematic representation of R67 DHFR shown as surface and ligands shown as sticks in the active site pore. A subset of the active site is shown in the enlarged version of the ligands and some active site residues. (B) Classical free-energy profile for the hydride transfer reaction catalyzed by R67 DHFR at different temperatures. The error bars are shown as highlighted regions around the solid lines. (C) Computed H/D KIEs for the R67 catalyzed hydride transfer reaction. Red and green points represent calculated KIEs in the enzyme and gas phase (with AM1-SRP parameterization), respectively, while blue points are experimental, KIEs. (D) Distribution of distances calculated between donor and acceptor atoms from ground state (GS) and TS trajectories. Colors: black, red, green, blue, and orange represent energies at temperatures 278, 288, 298, 308, and 318 K, respectively. The color-temperature notation also applies to (B).

Figure 2
Figure 2

RDF between DHF N5 and water oxygen for (A) GS and (B) TS trajectories. Colors: black, red, green, blue, and orange in (A,B) represent RDF at temperatures 278, 288, 298, 308, and 318 K, respectively. (C) Correlation between temperature and water count in GS and TS trajectories colored tan and cyan, respectively. The correlation coefficient was calculated using linear regression analysis.

Figure 3
Figure 3

(A) RMSF of Cα of R67 at different temperatures. The top and bottom panels represent RMSF at GS and TS, respectively. (B) Schematic representation of R67 DHFR shown as cartoons and substrate-cofactor shown as sticks in the active site pore. The red color in the cartoon representation depicts highly flexible regions that correspond to high RMSF values. The roman numbers designate the number of the monomers in R67 DHFR.

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