α-Secondary Isotope Effects as Probes of “Tunneling-Ready” Configurations in Enzymatic H-Tunneling:  Insight from Environmentally Coupled Tunneling Models

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

doi:10.1021/ja0614619

Cited by 66 publications

(169 citation statements)

References 48 publications

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“…One of the crucial findings from these studies is that the 1°KIE of MR is highly temperature-dependent (13). These data, coupled with a recent study showing that the 2°KIE is also exalted and consistent with preorganization in MR (42), have led us to describe the reaction within the context of modern environmentally coupled models of H-tunneling (12,20,43,44) such that the enzyme requires a promoting motion to move the nicotinamide C4-H sufficiently close to the FMN N5 atom to facilitate tunneling. Herein, we further this study by measuring the simultaneous temperature and pressure dependencies of the 1°KIE in this reaction.…”

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confidence: 54%

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Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects

Hay

Sutcliffe

Scrutton

2007

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

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193

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Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar ‫؍‬ 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 s ؊1 at 25°C) and deuterium (12 to 31 s ؊1 at 25°C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25°C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999) Can J Chem 77:1085-1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of ''barrier engineering'' by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions.flavoprotein ͉ hydrogen tunneling ͉ morphinone reductase ͉ pressure dependence ͉ stopped flow E nzymes are efficient catalysts that can achieve rate enhancements of up to 10 21 over the uncatalyzed reaction rate (1). Our quest to understand the physical basis of this catalytic power, which is pivotal to our understanding of biological reactions and our exploitation of enzymes in chemical, biomedical, and biotechnological processes, is challenging and has involved sustained and intensive research efforts for Ͼ100 years (for reviews see, e.g., refs. 2-6). Recent experimental studies employing the temperature dependence of kinetic isotope effects (KIEs) have emphasized the role of quantum mechanical tunneling in enzymatic hydrogen-transfer (H-transfer) reactions (7-18). These data, which can be explained neither by conventional transition state theory (TST) nor by the Bell tunneling correction to TST (19), are in agreement with environmentally coupled models of hydrogen tunneling (H-tunneling) (12,20,21) and can be simulated computationally (22-30) within the framework of modern TST (31). In the Kuznetsov and Ulstrup environmentally coupled model of H-tunneling (20), as used quantitatively by Klinman and coworkers (32), temperatureindependent KIEs arise from Marcus-like (33) vibrations (collective thermally equilibrated motions) that lead to degenerate reactant and product states, whereas temperature-dependent KIEs arise from ''gating motions'' (motion along the reaction coordinate) that enhance the probability of tunneling at this configuration by ...

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“…This reaction is directly observed in a rapid-mixing stopped-flow instrument and is kinetically resolved from steps involving coenzyme binding and formation of an enzyme-NADH charge-transfer (CT) complex, and the observed KIE is essentially the intrinsic KIE (13,41,42). The reductive half-reaction of this enzyme has been extensively characterized (13,41,42).…”

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confidence: 99%

Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects

Hay

Sutcliffe

Scrutton

2007

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

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193

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“…The term tunneling and coupled motion has been applied to ADHs and other systems that exhibit 2°KIEs outside the semiclassical range (12,28,37,38), as well as reactions that violate the Rule of the Geometric Mean, as evidenced by inflated mSSEs (1,18,21). The term tunneling and coupled motion appears to be used for both abnormalities in 2°KIEs.…”

Section: Resultsmentioning

confidence: 99%

“…This set guided our development of a more complete empirical model that explains all the apparently contradictory findings regarding the ADH reaction. This model implies a "tunneling ready state" (TRS), which is a special case of the more general TS (28). The TRS is the ensemble of states from which H tunneling occurs and like the general case of TS is a saddle point along a coordinate that represents the motions that bring the system from the ground state to the TRS.…”

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“…The TRS is the ensemble of states from which H tunneling occurs and like the general case of TS is a saddle point along a coordinate that represents the motions that bring the system from the ground state to the TRS. The present approach uses the framework of Marcus-like models of hydrogen tunneling (1,4,(28)(29)(30)(31)(32) and identifies some of the principle components of the collective reaction coordinate. 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.…”

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Elusive transition state of alcohol dehydrogenase unveiled

Roston

Kohen

2010

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

154

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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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Kinetic Isotope Effects in Enzymes

Kohen

Roston

Stojković

et al. 2010

Encyclopedia of Analytical Chemistry

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Kinetic isotope effects (KIEs) have progressed to become one of the most useful analytical techniques to study enzymatic reactions and can answer a variety of questions ranging from the basic mechanism of a reaction to the structure of a transition state (TS) and the role of quantum mechanical tunneling. Here, we briefly discuss the development of the theory behind KIEs with an emphasis on how it guides the interpretation of experimental data. We then present some of the instrumentation and techniques commonly used for KIE experiments, followed by a number of different examples from the enzymology literature wherein KIEs have been used to probe chemical transformations, with an emphasis on the effects of quantum mechanical tunneling on KIEs.

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α-Secondary Isotope Effects as Probes of “Tunneling-Ready” Configurations in Enzymatic H-Tunneling: Insight from Environmentally Coupled Tunneling Models

Cited by 66 publications

References 48 publications

Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects

Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects

Elusive transition state of alcohol dehydrogenase unveiled

Kinetic Isotope Effects in Enzymes

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