Small Temperature Dependence of the Kinetic Isotope Effect for the Hydride Transfer Reaction Catalyzed by Escherichia coli Dihydrofolate Reductase

Abstract

The H/D primary kinetic isotope effect (KIE) for the hydride transfer reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) is calculated as a function of temperature employing ensemble-averaged variational transition-state theory with multidimensional tunneling. The calculated KIEs display only a small temperature dependence over the temperature range of 5 to 45 °C. We identify two key features that contribute to canceling most of the temperature dependence of the KIE that would be expected on the basis of simpler models. Related issues such as the isotope effects on Arrhenius preexponential factors, large differences between free energies of activation and Arrhenius activation energy, and fluctuations of effective barriers are also discussed. This paper presents a theoretical explanation of the unusual temperature dependence 1 of the kinetic isotope effect (KIE) for the hydride transfer chemical step of the reaction catalyzed by E. coli dihydrofolate reductase (ecDHFR). The explanation identifies two general features that may be important in interpreting enzymatic kinetic isotope effects more generally.Many enzyme reactions involve hydron transfer (transfer of H + , H − , or H • ). Such reactions have significant contributions from zero-point energy and tunneling; KIEs have been extensively used to study these effects. [1][2][3][4][5][6][7] Surprisingly, a number of enzymes have been found to display almost temperature-independent KIEs, 1-4 which are contrary to experience with small-molecule chemistry or simple tunneling models. Recent multidimensional tunneling calculations have been successfully used to study tunneling effects on enzymatic KIEs, but the most accurate methods have so far been applied only at a single temperature. 8 DHFR catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7, with the key chemical step being the transfer of a hydride ion from the nicotinamide ring of the cofactor nicotinamide adinine dinucleotide phosphate (NADPH). At pH ≅ 7, product release is partly rate-limiting 9 and the H/D kinetic isotope effect is about 1.1-1.3, but the intrinsic KIE on the hydride transfer step is >3. 10 Furthermore, at 25 °C, the phenomenological free energy of activation derived from the rate constant 9 by using © 2005 American Chemical Society HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript transition-state theory is 13.4 kcal/mol, 7 and the free energy of reaction calculated from the experimental equilibrium constant 9 is −4.4 kcal/mol. 7Here, we report a dynamical simulation of the ecDHFR system using a combined quantum mechanical and molecular mechanical (QM/MM) approach. As depicted in Figure 1, the reactive fragment that involves transferring a hydride ion from the C4 position of NADPH (the cofactor) to the C6 in N5-preprotonated DHF (the substrate) to form THF (the product) is treated quantum mechanically. The hydride transfer KIEs at 5 and 45 °C and 1 atm are calculated using ensemble-averaged variational transition-stat...

Dynamics and dissipation in enzyme catalysis

“…For example, Neria and Karplus (32) used transmission coefficient calculations and constrained molecular dynamics (MD) trajectories to determine that the protein environment in triosephosphate isomerase (TIM) is essentially rigid (i.e., dynamically unresponsive) on the timescale of the intrinsic reaction dynamics; this finding is consistent with the lack of longlengthscale dynamical correlations reported in the current study. Furthermore, Truhlar and coworkers (33,34) and Karplus and Cui (35) both demonstrated that quasi-classical tunneling coefficients for hydrogen transfer evaluated at instantaneous enzyme configurations in the transition state region fluctuate significantly with donor-acceptor motions and other local active-site vibrations, which is likely consistent with the direct observation of short-lengthscale dynamical correlations reported here. However, by using quantized molecular dynamics to sample the ensemble of reactive trajectories in DHFR catalysis and to perform nonequilibrium ensemble averages that directly probe dynamical correlation, we provide a framework for strengthening and generalizing these earlier analyses.…”

Effects of a Distal Mutation on Active Site Chemistry

“…While studying ecDHFR for example, Garcia-Viloca et al (7) predicted a 2° KIE (which is a sensitive probe of the reaction's potential surface and was later confirmed experimentally (31)); Pu et al (8) could reproduce the trends of temperature dependence for 1° KIEs, and Agarwal et al (28) could reproduce and predict several experimental data. All these studies could address the role of protein motion and other mechanistic features that are not directly accessible by experimental studies.…”

“…Furthermore the essential role of DHFR in DNA synthesis and in a variety of anabolic pathways makes it a common target for antiproliferative therapeutics. Due to its biological and pharmacological importance, and it being a small monomeric enzyme, DHFR has been the subject of intensive structural and kinetic investigation over many years, serving as a paradigm of enzymatic systems in many experimental and theoretical studies (1)(2)(3)(4)(5)(6)(7)(8). † This work was supported by NIH R01 GM65368-01 and NSF CHE-0133117 to A.K.…”

Unraveling the role of protein dynamics in dihydrofolate reductase catalysis

“…Whereas some authors postulate dynamics as a key driving force in catalysis (1)(2)(3)(4), others have performed analyses showing activation free-energy reduction, which is an equilibrium property, to be the source of catalysis (6)(7)(8)(9)(10)(11)(12)(13)(14). Enzyme reactions, and particularly their dynamics, present formidable challenges for study, and progress requires a combination of theoretical, experimental, and computational approaches (5,(15)(16)(17)(18) (20). The physical steps of ligand association and dissociation have been shown to depend on movements between these two conformations (20,21).…”