Nonperfect Synchronization of Reaction Center Rehybridization in the Transition State of the Hydride Transfer Catalyzed by Dihydrofolate Reductase

Pu, Jingzhi; Ma, Shuhua; Garcia‐Viloca, Mireia; Gao, Jiali; Truhlar, Donald G.; Kohen, Amnon

doi:10.1021/ja054170t

Cited by 57 publications

(120 citation statements)

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“…This empirically parameterized model quantitatively replicates all of the 2°KIEs in the forward and reverse directions, as well as their mSSEs, and the LFER findings. The model also indicates an asynchronized hybridization for the donor and acceptor carbons, which was first identified for dihydrofolate reductase (DHFR) by QM/MM calculations (33). Finally, in accordance with the ensemble-averaged variational transition state theory (VTST) results from Truhlar, Gao, and co-workers (24,25), the model supports the recently articulated hypothesis that the inflated mSSEs reported for many ADHs since 1989 result from deflated 2°D/T KIEs because of the shorter donor-acceptor distance (DAD) required for D transfer (1,34).…”

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confidence: 65%

“…3 mirrors the VTST treatment (39) that has been quite successful in modeling enzymatic reactions that involve H tunneling (24,25,33). In these models, the enzyme facilitates tunneling of the transferred particle by creating energetic degeneracy between a donor and acceptor state.…”

Section: Resultsmentioning

confidence: 99%

“…Traditionally, models expected the magnitude of 2°KIEs to vary between unity for a reactant-like TS and the EIE for a product-like TS (see ref. 33 for a thorough discussion), but quantum mechanical tunneling and 1°-2°coupled motion have given experimental results that elude such simplistic interpretations. The 2°H/T KIE measured here for the reduction of benzaldehyde by NADH clearly escapes interpretation by semiclassical models.…”

Section: Resultsmentioning

confidence: 99%

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Elusive transition state of alcohol dehydrogenase unveiled

Roston

Kohen

2010

Proc. Natl. Acad. Sci. U.S.A.

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For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (>1) 2°kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (<1), this KIE eludes the traditional interpretation of 2°KIEs. It does, however, enable the development of a comprehensive model for the "tunneling ready state" (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate.hydrogen tunneling | enzyme kinetic | secondary isotope effect | Swain-Schaad E nzymes enhance the rates of chemical reactions by many orders of magnitude, and extensive studies have uncovered many aspects of how they do so. The prevailing theory that enzymes stabilize the transition state (TS) relative to the reactants explains many phenomena, and a great deal of contemporary research focuses on how enzymes stabilize the TS. An unfortunate hindrance to developing an understanding of how enzymes stabilize the TS lies in the enigmatic nature of the TS. Many enzymatic hydrogen (proton, hydrogen, or hydride) transfers, for example, occur by quantum mechanical tunneling, where a particle passes through an energy barrier because of its wave properties (1-6).Yeast alcohol dehydrogenase (yADH) serves as an excellent model system for enzyme-catalyzed H transfer because unlike many other enzymes, the chemical step, oxidation of a primary alcohol to an aldehyde by NAD þ , is rate-limiting with aromatic substrates (7). Furthermore, the thermodynamics of the reaction allow for examination of both the forward (alcohol to aldehyde) and reverse (aldehyde to alcohol) reactions under similar conditions (8). This type of nicotinamide-dependent redox reaction appears ubiquitously in biology, and a detailed picture of the TS of such reactions may facilitate many medical and industrial applications.Intriguingly, decades of experiments on the reaction catalyzed by yADH have returned what appear to be contradictory results, some suggesting an early TS, whereas others point to a late TS (7-10). In pioneering studies, Klinman measured linear fr...

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confidence: 65%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Elusive transition state of alcohol dehydrogenase unveiled

Roston

Kohen

2010

Proc. Natl. Acad. Sci. U.S.A.

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154

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“…The lack of coupled motion for both enzymes agrees with a similar nature of the H-transfer mechanism, but reduced 2° KIEs indicate some differences in the system's vibrational states, which could be in agreement with different rearrangements of the potential surface close to the transition state. Interestingly, a more rigorous MM/QM study of the 2° KIEs for the wild type ecDHFR indicated non-synchronized hybridization of the donor and acceptor carbons and slightly associative transition state (91). A similar study of the G121V mutant is under way, and it's findings may shed light on the differences between the transition state ensembles of the wild type and G121V ecDHFRs.…”

Section: "Marcus Like" Models (Environmentally Coupled Tunneling)mentioning

confidence: 86%

Effects of a Distal Mutation on Active Site Chemistry

et al. 2006

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Previous studies of E. coli dihydrofolate reductase (ecDHFR) have demonstrated that residue G121, which is 19Å away from the catalytic center, is involved in catalysis and long distance dynamical motions were implied. Specifically, the ecDHFR mutant G121V has been extensively studied by various experimental and theoretical tools, and the mutation's effect on kinetic, structural, and dynamical features of the enzyme explored. The current work examined the effect of this mutation on the physical nature of the catalyzed hydride transfer step by means of intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters as described before for the wild type ecDHFR (Sikorski et al. 2004, J. Am. Chem. Soc., 126, 4778-4779). The temperature dependence of initial velocities was used to estimate activation parameters. Isotope effects on the preexponential Arrhenius factors, and the activation energy could be rationalized by an environmentally coupled hydrogen tunneling model, similar to the one used for the wild type enzyme. Yet, in contrast to the wild type, fluctuations of the donor-acceptor distance were now required. Secondary (2°) KIEs were also measured for both H and D transfer and, as in the case of the wild type enzyme, no coupled motion was detected. Despite these similarities, the reduced rates, the slightly inflated 1° KIEs and their temperature dependence, together with relatively deflated 2° KIEs, indicate that the potential surface prearrangement was not as ideal as for the wild type enzyme. These findings support theoretical studies suggesting that the G121V mutation lead to a different conformational ensemble of reactive states and less effective rearrangement of the potential surface, but has only small effect on H-tunneling.Dihydrofolate reductase from Escherichia coli (ecDHFR) is a small monomeric enzyme (18 kDa) that consists of eight-stranded β-sheet and four α-helices connected with several loop regions. It catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7, with the concomitant oxidation of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) to NADP + , in which a hydride is stereospecifically transferred from the pro-R C4 position of the nicotinamide ring to the si face of the C6 position of pterin. This enzyme helps maintain intracellular pools of THF used in the biosynthesis of purine nucleotides and some amino acids. 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. The complete kinetic scheme for wild type ecDHFR was derived from equilibrium binding, steady-sta...

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“…[512][513][514] Alternatively, for H transfer, when the donor and acceptor experience changes of the hybridization states, recent studies emphasized the usefulness of employing the change of one or both hybridization states as the DRC. 408 Any combination of bond distances, bond angles, or torsion angles is called a valence coordinate, whereas quantities based on diabatic potentials or electrostatic fields that depend on solvent or bath coordinates are often called collective solvent coordinates. Valence coordinates are also called geometrical coordinates.…”

Section: Ensemble-averaged Variational Transition State Theory With Mmentioning

confidence: 99%