Comp Chem Research Developments

Archive of Comp Chem Research News
December 11, 2003
    DHFR Kinetic Isotope Effects

    Dihydrofolate reductase (DHFR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF). DHFR maintains intracellular pools of tetrahydrofolate and is essential for biosynthesis; hence it is a target for anticancer and antibacterial drugs, and its clinical importance has led to numerous experimental and theoretical studies of its catalytic mechanism. The overall reaction is rate limited at high pH by a hydride transfer step. To provide a deeper understanding of DHFR catalysis, postdoctoral associate Mireia Garcia-Viloca and Professors Jiali Gao and Donald Truhlar used combined quantum mechanical and molecular mechanical calculations to study the effects of the enzyme electric field on the molecular polarization of the cofactor, NADPH, and the substrate for the Michaelis complex, the transition state, and the product-enzyme complex. The method can be useful for the rational drug design of inhibitors to DHFR. Figure a shows the electron density difference plot for 5-protonated dihydrofolate substrate in the active center of the enzyme. Figure b is the same for the tetrahydrofolate product. Blue contours represent regions where there is a depletion of electron density, and red contours indicate areas where there is a gain, upon transferring the substrate from the gas phase into the active site.

    With these insights, a potential energy surface was modeled, and the reaction rate and kinetic isotope effects were calculated by ensemble-averaged variational transition state theory with multidimensional tunneling. A primary kinetic isotope effect (ratio of the rate constant for transferring hydride to that for deuteride) of 2.8 has been obtained, in good agreement with the experimentally determined value of 3.0. The primary KIE is mainly a consequence of the quantization of bound vibrations. In contrast, the secondary kinetic isotope effect, corresponding to deuteration at a nonreactive site, is predicted to have a value of 1.13, which is almost entirely due to dynamical effects on the reaction coordinate, especially tunneling. When this was calculated, there was no experiment available, but it has now been confirmed by measurements carried out by Professor Amnon Kohen of the University of Iowa, making it the first ever successful calculation of such an effect prior to its being measured.

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