Direct Analysis of Donor−Acceptor Distance and Relationship to Isotope Effects and the Force Constant for Barrier Compression in Enzymatic H-Tunneling Reactions

Pudney, Christopher R.; Johannissen, Linus O.; Sutcliffe, Michael J.; Hay, Sam; Scrutton, Nigel S.

doi:10.1021/ja1048048

Cited by 75 publications

(162 citation statements)

References 55 publications

(153 reference statements)

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“…Heavy isotope labeling is expected to reduce the frequencies of harmonic bond vibrations, while having minimal effect on electrostatics. In enzymes where vibrations lead to active site 'compression' such as the transient reduction in hydrogen donor-acceptor atoms, which has been suggested for OYEs such as PETNR, 1,25,[39][40][41] then perturbation of such vibrations can lead to altered reaction kinetics by altering the probability of transition state formation and/or the rate of transition state recrossing. 17,18,42 Slower dynamics will also be affected by mass perturbation, which may lead to altered substrate/product binding and release kinetics.…”

Section: Resultsmentioning

confidence: 99%

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer

et al. 2016

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'Heavy' (isotopically-labeled) enzyme isotope effects offer a direct experimental probe of the role of protein vibrations on enzyme-catalyzed reactions. Here we have developed a strategy to generate isotopologues of the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) where the protein and/or intrinsic flavin mononucleotide (FMN) cofactor are isotopically labeled with 2 H, 15 N and 13 C. Both the protein and cofactor contribute to the enzyme isotope effect on the reductive hydride transfer reaction, but their contributions are not additive and may partially cancel each other out. However, the isotope effect specifically arising from the FMN suggests that vibrations local to the active site play a role in the hydride transfer chemistry, while the protein-only 'heavy enzyme' effect demonstrates that protein vibrations contribute to catalysis in PETNR. In all cases, enthalpy-entropy compensation plays a major role in minimizing the magnitude of 'heavy enzyme' isotope effects. Fluorescence lifetime measurements of the intrinsic flavin mononucleotide show marked differences between 'light' and 'heavy' enzymes on the ns-ps timescale, suggesting relevant timescale(s) for those vibrations implicated in the 'heavy enzyme' isotope effect on the PETNR reaction.

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Section: Resultsmentioning

confidence: 99%

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer

et al. 2016

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“…This can be accomplished in two distinct ways. The reduced distance can be reached via a local, picosecond-nanosecond timescale distance sampling contribution to the reorganization barrier (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Evidence for distance sampling has been obtained repeatedly from highly temperature-dependent kinetic isotope effects and has been discussed extensively in the literature (refs.…”

Section: Discussionmentioning

confidence: 99%

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

Nagel

Dong

Bahnson

et al. 2011

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

154

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A growing body of data supports a role for protein motion in enzyme catalysis. In particular, the ability of enzymes to sample catalytically relevant conformational substates has been invoked to model kinetic and spectroscopic data. However, direct experimental links between rapidly interconverting conformations and the chemical steps of catalysis remain rare. We report here on the kinetic analysis and characterization of the hydride transfer step catalyzed by a series of mutant thermophilic alcohol dehydrogenases (ht-ADH), presenting evidence for Arrhenius prefactor values that become enormously elevated above an expected value of approximately 10 13 s −1 when the enzyme operates below its optimal temperature range. Restoration of normal Arrhenius behavior in the ht-ADH reaction occurs at elevated temperatures. A simple model, in which reduced temperature alters the ability of the ht-ADH variants to sample the catalytically relevant region of conformational space, can reproduce the available data. These findings indicate an impaired landscape that has been generated by the combined condition of reduced temperature and mutation at a single, active-site hydrophobic side chain. The broader implication is that optimal enzyme function requires the maintenance of a relatively smooth landscape that minimizes low energy traps.A complete understanding of the origins of the remarkable rate acceleration and specificity achieved by enzymes remains elusive. Increasingly, studies on the fundamental basis for enzymatic catalysis have focused on the requirement for a range of protein motions in order to achieve efficient enzyme catalysis. Among these, evidence for a class of motion termed conformational sampling (or preorganization) has begun to accumulate recently (cf. refs. 1-4). The impact of such motions can be seen either in the formation of an enzyme-substrate complex or in its subsequent conversion to product.For example, a recent study of adenylate kinase implicates a temperature-dependent preequilibrium among conformers that are either "competent" or "incompetent" toward ligand binding (5). In the context of catalysis, studies of single-enzyme molecules indicate multiple, slowly interconverting conformers that lead to product with different rate constants (6-8). Extensive work on dihydrofolate reductase has shown the role of slow loop closures that occur along the reaction coordinate with the same (millsecond) time constants as catalysis (9, 10).Although computational studies also suggest a role for rapid conformational sampling in enzyme reactions (11, 12), demonstrating the direct link of these motions to catalysis is quite challenging. In this study, we use the thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) as a system to examine the presence of rapidly interconverting protein substates during catalysis. The ht-ADH catalyzes the transfer of a hydride equivalent from an alcohol donor to an NAD þ cofactor, and previous studies have shown that ht-ADH carries out its reaction via quantum t...

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“…The finding that the KIEs, including the isotope effects on the Arrhenius pre-factor (that indicate the tunneling in the reaction, see below) observed in the presence of added cations differ significantly from the KIEs observed in the absence of cations presented a direct evidence of an influence of cations on the PCET process. Within the framework of the Marcus-like tunneling model [40 -60], the observed changes in KIE can be viewed as a consequence of changes in both barrier height and width due to the changes both in the donorÀacceptor distance for Htransfer and the force constant of compressive modes coincident with the reaction coordinate [55] [56] [59] [60], resulting from interactions of ionic environment with the activated complex in the reaction (see also below). The observations are: in the Na þ concentration range, the prevailing reaction channel probably is that involving the cation in a proximity of the activated complex in the PCET reaction, although the cation in the solvent cage is neither the redox partner nor involved in the proton transfer in the reaction.…”

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confidence: 99%

“…It has also been shown [57] that a modification of the model can be applied to a system that does not involve a protein environment. Scrutton and co-workers [59] [60] have suggested that compressive modes (the donorÀacceptor distance fluctuations coincident and coupled with the reaction coordinate, promoting modes) may be a feature of both solution and enzyme systems.…”

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confidence: 99%

Ions Can Move a Proton‐Coupled Electron‐Transfer Reaction into Tunneling Regime

Brala

Pilepić

Sajenko

et al. 2011

Helvetica Chimica Acta

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The effects of charged species on proton-coupled electron-transfer (PCET) reaction should be of significance for understanding/application of important chemical and biological PCET systems. Such species can be found in proximity of activated complex in a PCET reaction, although they are not involved in the charge transfer process. Reported here is the first study of the above-mentioned effects. Here, the effects of Na , 5.5 (0.9) (at 0.001m Ca 2þ ), and 7.9 (2.8) (at 0.001m Mg 2þ ) kJ mol À1 ; e) nonlinear proton inventory in reaction. In the H 2 O/dioxane 1 : 1, the observed KIE is 7.8 and 4.4 in the absence and in the presence of 0.1m K þ , respectively, and A H /A D ¼ 0.14 (0.03). The changes when cations are present in the reaction are explained in terms of termolecular encounter complex consisting of redox partners, and the cation where the cation can be found in a near proximity of the reaction-activated complex thus influencing the proton/electron double tunneling event in the PCET process. A molecule of H 2 O is involved in the transition state. The resulting configuration is more rigid and more appropriate for efficient tunneling with Na þ or K þ (extensive tunneling observed), i.e., there is more precise organized H transfer coordinate than in the case of Ca 2þ and Mg 2þ (moderate tunneling observed) in the reaction.Introduction. -Proton-coupled electron-transfer (PCET) reactions are of crucial importance in a wide range of biological, biochemical, and chemical systems [1 -16], including those related to artificial photosynthesis and solar fuels [4 -8], and nanostructures and interfaces [14] [15]. It is recognized now that many processes in biology would not be possible without the coupling of proton and electron motion [3] [5]. Within a unified theoretical framework, both sequential electron transfer, followed by proton transfer (ET/PT) or vice versa (PT/ET), and the concerted transfer of these particles with conspicuous quantum-mechanical character could be viewed as PCET reactions [16]. However, the concerted transfer of a proton and an electron should be of central importance in the issue; here, the single chemical reaction step and

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Direct Analysis of Donor−Acceptor Distance and Relationship to Isotope Effects and the Force Constant for Barrier Compression in Enzymatic H-Tunneling Reactions

Cited by 75 publications

References 55 publications

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

Ions Can Move a Proton‐Coupled Electron‐Transfer Reaction into Tunneling Regime

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