Are Environmentally Coupled Enzymatic Hydrogen Tunneling Reactions Influenced by Changes in Solution Viscosity?

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

doi:10.1002/anie.200704484

Cited by 34 publications

(42 citation statements)

References 42 publications

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“…First, we re-analyze the dependence of k obs on glycerol concentration (data taken from ref. [27]) in terms of the solvent dielectric. 2 Additionally, we also measure the rate of the RHR of MR in 10 % and 20 % v/v ethanol.…”

Section: Solvent Dielectricmentioning

confidence: 99%

“…In combination, this has led us to describe the hydride transfer (denoted H-transfer) within the context of modern environmentally coupled models of H-tunnelling, [19][20][21]26] such that the enzyme requires a promoting motion to move the nicotinamide C4ÀH sufficiently close to the FMN N5 atom to facilitate H-transfer by tunnelling. Recently, we have shown that the rate of the H-transfer reaction in MR is insensitive to changes in solution viscosity, [27] suggesting that this putative promoting motion, like that for aromatic amine dehydrogenase, [11,28] may be localized at the active site of the enzyme (c.f. the case argued for long-range coupled motions in for example, alcohol dehydrogenase, [29,30] The reductive half-reaction of morphinone reductase involves a hydride transfer from enzyme-bound b-nicotinamide adenine dinucleotide (NADH) to a flavin mononucleotide (FMN).…”

Section: Introductionmentioning

confidence: 99%

“…More evidence is required to identify the origin of the putative promoting motion in MR, but there is no evidence for a long-range network of coupled motions in this enzyme. [27] In fact, it is quite possible that the putative "promoting motion" in MR is simply one or more inherent vibrations within either the NADH nicotinamide and/or FMN isoalloxazine moieties. 4 The crystal structure of the MRÀ NADH 4 complex (Figure 4) shows water within the active site but little water is found directly around either the nicotinamide or isoalloxazine.…”

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confidence: 99%

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Solvent as a Probe of Active Site Motion and Chemistry during the Hydrogen Tunnelling Reaction in Morphinone Reductase

et al. 2008

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The reductive half-reaction of morphinone reductase involves a hydride transfer from enzyme-bound beta-nicotinamide adenine dinucleotide (NADH) to a flavin mononucleotide (FMN). We have previously demonstrated that this step proceeds via a quantum mechanical tunnelling mechanism. Herein, we probe the effect of the solvent on the active site chemistry. The pK(a) of the reduced FMN N1 is 7.4+/-0.7, based on the pH-dependence of the FMN midpoint potential. We rule out that protonation of the reduced FMN N1 is coupled to the preceding H-transfer as both the rate and temperature-dependence of the reaction are insensitive to changes in solution pH above and below this pK(a). Further, the solvent kinetic isotope effect is approximately 1.0 and both the 1 degrees and 2 degrees KIEs are insensitive to solution pH. The effect of the solvent's dielectric constant is investigated and the rate of H-transfer is found to be unaffected by changes in the dielectric constant between approximately 60 and 80. We suggest that, while there is crystallographic evidence for some water in the active site, the putative promoting motion involved in the H-tunnelling reaction is insensitive to such changes.

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Section: Solvent Dielectricmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Solvent as a Probe of Active Site Motion and Chemistry during the Hydrogen Tunnelling Reaction in Morphinone Reductase

et al. 2008

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“…For studies of the temperature dependence of rate constants, data were fitted to the Eyring equation. For viscosity studies, glycerol solutions were prepared by weight and calculation of solution viscosity was as described (24). The effect of viscosity on the observed rate was fitted to Equation 1 (25,26) to describe the contribution of the protein friction to the total friction of the system,…”

Section: Methodsmentioning

confidence: 99%

Mutagenesis Alters the Catalytic Mechanism of the Light-driven Enzyme Protochlorophyllide Oxidoreductase

Menon

Davison

Hunter

et al. 2010

Journal of Biological Chemistry

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The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes an essential step in the synthesis of the most abundant pigment on Earth, chlorophyll. This unique reaction involves the sequential addition of a hydride and proton across the C17‫؍‬C18 double bond of protochlorophyllide (Pchlide) by dynamically coupled quantum tunneling and is an important model system for studying the mechanism of hydrogen transfer reactions. In the present work, we have combined site-directed mutagenesis studies with a variety of sensitive spectroscopic and kinetic measurements to provide new insights into the mechanistic role of three universally conserved Cys residues in POR. We show that mutation of Cys-226 dramatically alters the catalytic mechanism of the enzyme. In contrast to wild-type POR, the characteristic charge-transfer intermediate, formed upon hydride transfer from NADPH to the C17 position of Pchlide, is absent in C226S variant enzymes. This suggests a concerted hydrogen transfer mechanism where proton transfer only is rate-limiting. Moreover, Pchlide reduction does not require the network of solvent-coupled conformational changes that play a key role in the proton transfer step of wild-type POR. We conclude that this globally important enzyme is finely tuned to facilitate efficient photochemistry, and the removal of a key interaction with Pchlide in the C226S variants significantly affects the local active site structure in POR, resulting in a shorter donoracceptor distance for proton transfer.The light-activated enzyme protochlorophyllide oxidoreductase (POR) 3 catalyzes the reduction of protochlorophyllide (Pchlide) (Fig. 1A), a key reaction in the chlorophyll biosynthetic pathway that triggers a profound transformation in plant development (1-3). Although the reaction can also be catalyzed by a light-independent Pchlide reductase in non-flowering land plants, algae, and cyanobacteria, higher plants only contain POR and are therefore completely reliant on light for the greening process (1, 2). The light dependence of POR presents a unique opportunity to study catalysis at low temperatures and on ultrafast timescales, both of which are usually inaccessible for the majority of enzymes (1). Recent advances in our understanding of the catalytic mechanism of POR illustrate why it is an important generic model for studying enzyme catalysis and reaction dynamics (4 -12).Low temperature spectroscopy indicates that the catalytic cycle of the enzyme comprises an initial light-driven reaction that involves hydride transfer from the pro-S face of NADPH to the C17 of Pchlide to form a charge-transfer complex (4, 5). This facilitates the subsequent protonation of the C18 position of the Pchlide molecule from a conserved Tyr residue during the first of the dark reactions (5-7). By using laser photoexcitation studies, we have shown that these two sequential enzymatic H-transfer reactions occur by quantum mechanical tunneling (8). Proton tunneling is coupled to promoting motions in the enzyme that facilitate the re...

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“…Significant changes in the magnitude of an enzymatic KIE through, e.g., environmental perturbations (Hay et al , 2008b or mutagenesis (Pudney et al 2007) will generally occur, in part or wholly, due to structural perturbation of the active site. Care must then be taken when interpreting such data.…”

Section: Experimental Evidence For H-tunneling In Enzymesmentioning

confidence: 99%

H-transfers in Photosystem II: what can we learn from recent lessons in the enzyme community?

Hay

Scrutton

2008

Photosynth Res

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Over the last 10 years, studies of enzyme systems have demonstrated that, in many cases, H-transfers occur by a quantum mechanical tunneling mechanism analogous to long-range electron transfer. H-transfer reactions can be described by an extension of Marcus theory and, by substituting hydrogen with deuterium (or even tritium), it is possible to explore this theory in new ways by employing kinetic isotope effects. Because hydrogen has a relatively short deBroglie wavelength, H-transfers are controlled by the width of the reaction barrier. By coupling protein dynamics to the reaction coordinate, enzymes have the potential ability to facilitate more efficient H-tunneling by modulating barrier properties. In this review, we describe recent advances in both experimental and theoretical studies of enzymatic H-transfer, in particular the role of protein dynamics or promoting motions. We then discuss possible consequences with regard to tyrosine oxidation/reduction kinetics in Photosystem II.

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Are Environmentally Coupled Enzymatic Hydrogen Tunneling Reactions Influenced by Changes in Solution Viscosity?

Cited by 34 publications

References 42 publications

Solvent as a Probe of Active Site Motion and Chemistry during the Hydrogen Tunnelling Reaction in Morphinone Reductase

Solvent as a Probe of Active Site Motion and Chemistry during the Hydrogen Tunnelling Reaction in Morphinone Reductase

Mutagenesis Alters the Catalytic Mechanism of the Light-driven Enzyme Protochlorophyllide Oxidoreductase

H-transfers in Photosystem II: what can we learn from recent lessons in the enzyme community?

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