Reaction-Path Energetics and Kinetics of the Hydride Transfer Reaction Catalyzed by Dihydrofolate Reductase

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• 作者：Mireia Garcia-Viloca ; Donald G. Truhlar ; and Jiali Gao
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：November 25, 2003
• 年：2003
• 卷：42
• 期：46
• 页码：13558 - 13575
• 全文大小：370K
• 年卷期：v.42,no.46(November 25, 2003)
• ISSN：1520-4995

文摘

We have studied the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase(DHFR) and the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH); the substrate is5-protonated 7,8-dihydrofolate, and the product is tetrahydrofolate. The potential energy surface is modeledby a combined quantum mechanical-molecular mechanical (QM/MM) method employing Austin model1 (AM1) and a simple valence bond potential for 69 QM atoms and employing the CHARMM22 andTIP3P molecular mechanics force fields for the other 21 399 atoms; the QM and MM regions are joinedby two boundary atoms treated by the generalized hybrid orbital (GHO) method. All simulations arecarried out using periodic boundary conditions at neutral pH and 298 K. In stage 1, a reaction coordinateis defined as the difference between the breaking and forming bond distances to the hydride ion, and aquasithermodynamic free energy profile is calculated along this reaction coordinate. This calculation includesquantization effects on bound vibrations but not on the reaction coordinate, and it is used to locate thevariational transition state that defines a transition state ensemble. Then, the key interactions at the reactant,variational transition state, and product are analyzed in terms of both bond distances and electrostaticenergies. The results of both analyses support the conclusion derived from previous mutational studiesthat the M20 loop of DHFR makes an important contribution to the electrostatic stabilization of the hydridetransfer transition state. Third, transmission coefficients (including recrossing factors and multidimensionaltunneling) are calculated and averaged over the transition state ensemble. These averaged transmissioncoefficients, combined with the quasithermodynamic free energy profile determined in stage 1, allow usto calculate rate constants, phenomenological free energies of activation, and primary and secondary kineticisotope effects. A primary kinetic isotope effect (KIE) of 2.8 has been obtained, in good agreement withthe experimentally determined value of 3.0 and with the value 3.2 calculated previously. The primaryKIE is mainly a consequence of the quantization of bound vibrations. In contrast, the secondary KIE,with a value of 1.13, is almost entirely due to dynamical effects on the reaction coordinate, especiallytunneling.