Enzyme structure and dynamics affect hydrogen tunneling: The impact of a remote side chain (I553) in soybean lipoxygenase-1

Meyer, Matthew P.; Tomchick, Diana R.; Klinman, Judith P.

doi:10.1073/pnas.0710643105

Cited by 153 publications

(318 citation statements)

References 36 publications

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“…Mounting data for enzymes that transfer hydrogen by a tunneling mechanism have led to a model for enzyme catalysis that implicates at least two classes of protein motion (15,16,(25)(26)(27)(28)(29)(30). Tunneling has been modeled with an expanded Marcus-like equation that assigns the thermal reaction barrier to the environmental reorganization terms that produce transient degeneracy between reactant and product wells and lead to sampling of a range of donor-acceptor distances (31)(32)(33).…”

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.

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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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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.

Self Cite

154

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“…To assess tunneling contributions to catalysis, it is rare that a reaction in water can be compared with the same reaction catalyzed by an enzyme in sufficient detail (2,12). In the present study, we present overwhelming evidence demonstrating quantitatively differential NQE that contributes to catalysis in NAO: We found that the overall NQE has a greater contribution to lowering the classical barrier in the enzyme, and there is a larger difference in quantum effects between proton and deuteron transfer for the enzymatic reaction than the uncatalyzed one.…”

Section: Discussionmentioning

confidence: 99%

“…Although tantalizing, the relatively small difference in the observed KIEs is not sufficient to conclude that there is a greater tunneling contribution in the enzymatic process than that of the uncatalyzed reaction in water. A number of experimental and theoretical studies suggested that the extent of tunneling was the same in several enzymes as in model reactions in solution (2,9,10), whereas other experiments showing differential tunneling behaviors may be attributed to different reaction mechanisms (2,(11)(12)(13). Nuclear tunneling cannot be measured directly, and the best experimental diagnostic of tunneling is through measurement of kinetic isotope effects (14).…”

mentioning

confidence: 99%

Differential quantum tunneling contributions in nitroalkane oxidase catalyzed and the uncatalyzed proton transfer reaction

Major

Héroux

Orville

et al. 2009

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

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The proton transfer reaction between the substrate nitroethane and Asp-402 catalyzed by nitroalkane oxidase and the uncatalyzed process in water have been investigated using a path-integral free-energy perturbation method. Although the dominating effect in rate acceleration by the enzyme is the lowering of the quasiclassical free energy barrier, nuclear quantum effects also contribute to catalysis in nitroalkane oxidase. In particular, the overall nuclear quantum effects have greater contributions to lowering the classical barrier in the enzyme, and there is a larger difference in quantum effects between proton and deuteron transfer for the enzymatic reaction than that in water. Both experiment and computation show that primary KIEs are enhanced in the enzyme, and the computed Swain-Schaad exponent for the enzymatic reaction is exacerbated relative to that in the absence of the enzyme. In addition, the computed tunneling transmission coefficient is approximately three times greater for the enzyme reaction than the uncatalyzed reaction, and the origin of the difference may be attributed to a narrowing effect in the effective potentials for tunneling in the enzyme than that in aqueous solution.PI-FEP/UM simulations ͉ enzyme catalysis ͉ kinetic isotope effects ͉ X-ray structure A lthough the dominant factor in enzyme catalysis is the lowering of the quasiclassical free energy barrier of the enzymatic reaction in comparison with the uncatalyzed process (1-3), quantum mechanical tunneling has been recognized to also play a role in enzymatic hydrogen transfer reactions (2, 3). An intriguing, yet unanswered, question is whether enzymes have evolved to enhance tunneling to accelerate the reaction rate because quantum effects on rate acceleration are much smaller than hydrogen bonding and electrostatic stabilization of the transition state (1, 4, 5). Nevertheless, a small factor of two in rate enhancement can have important physiological impacts. Although it appears straightforward to address this question by comparing the enzymatic and the uncatalyzed reaction in solution, the difficulty is to design a model system that mimics exactly the same enzymatic reaction and mechanism. The present study examines the structure of nitroalkane oxidase (NAO) complex with nitroethane and kinetic isotope effects at the primary and secondary sites for the enzymatic and the uncatalyzed reaction. The computational findings are consistent with experimental data, suggesting that there is a differential tunneling effect for the proton transfer reaction in NAO and in water. Analysis of tunneling paths reveals that the enzyme reduces both the free energy of activation and the width of the effective potential, resulting in enhanced proton tunneling in the active site.The flavoenzyme NAO catalyzes the conversion of nitroalkanes to nitrite and aldehydes or ketones (Fig. S1) (6). The ␣-proton abstraction of the small substrate nitroethane by Asp-402 is rate-limiting, which is accelerated by a factor of 10 9 over the uncatalyzed reaction between n...

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“…The nature and magnitude of these forces remains an active area of research. [10][11][12][13][14][15] Enzymes have evolved to create elaborate active sites containing precise arrangements of hydrogen-bonding networks, electrostatic interactions, and functional group availability specifically aimed at increasing the reactivity of bound substrates. While the development of synthetic host-guest systems has not reached the level of enzyme specificity, the characteristics of each synthetic assembly, such as the size, shape, charge, and functional group availability, greatly influence the guest-binding characteristics and have led to remarkable and often unexpected reactivity.…”

Section: Introductionmentioning

confidence: 99%

Spin Equilibria in Monomeric Manganocenes: Solid-State Magnetic and EXAFS Studies

et al. 2009

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A water-soluble self-assembled supramolecular host molecule catalyzes the hydrolysis of orthoformates in basic solution. Comparison of the rate constants of the catalyzed and uncatalyzed reactions for hydrolysis displays rate accelerations of up to 3900 for tri-n-propyl orthoformate. Kinetic analysis shows that the mechanism of hydrolysis with the supramolecular host obeys the Michaelis-Menten model.Mechanistic studies, including 13 C-labeling experiments, revealed that the resting state of the catalytic system is the neutral substrate encapsulated in the host. Activation parameters for the k cat step of the reaction revealed that upon encapsulation in the assembly, the entropy of activation becomes more negative in contrast to the uncatalyzed reaction. Furthermore, solvent isotope effects reveal a normal k(H 2 O)/k(D 2 O) = 1.6, confirming that proton transfer is occurring in the transition state and is rate limiting in the catalyzed reaction. In comparison to the uncatalyzed reaction, which operates by an A-1 mechanism in which the decomposition of the protonated substrate is rate limiting, the encapsulated reaction proceeds through an A-S E 2 mechanism in which proton transfer from protonated water to the substrate is rate limiting.

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Enzyme structure and dynamics affect hydrogen tunneling: The impact of a remote side chain (I553) in soybean lipoxygenase-1

Cited by 153 publications

References 36 publications

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

Differential quantum tunneling contributions in nitroalkane oxidase catalyzed and the uncatalyzed proton transfer reaction

Spin Equilibria in Monomeric Manganocenes: Solid-State Magnetic and EXAFS Studies

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