Hydrostatic Pressure Studies Distinguish Global from Local Protein Motions in C−H Activation by Soybean Lipoxygenase‐1

Shi-sheng, HU; Cattin‐Ortolá, Jérôme; Munos, Jeffrey W.; Klinman, Judith P.

doi:10.1002/ange.201603592

Cited by 5 publications

(5 citation statements)

References 34 publications

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“…A related temperature−pressure perturbation study of DM SLO also highlights the distinctive features of this variant. 28,32 Unlike the pressure-induced shifted conformational landscape in WT and three single mutants (L546A, L754A and I553V), the conformational landscape of the enzyme−substrate complex of DM is more resistant to perturbations from elevated pressure, showing a consistently high energy barrier (E a = 8−9.9 kcal/mol) and slow catalytic efficiency (10 4 -fold decrease) relative to WT. At this juncture, a major unanswered question is the structural origin underlying the aggregated features of DM SLO.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Differences between experimental KIEs during the biomolecular reaction of enzyme with substrate (k cat /K m ) and the unimolecular turnover rate (k cat ) led to the proposal of perturbed conformational landscapes in selected mutants. 31 The further application of various biophysical approaches that include studies of increased hydrostatic pressure, 32 hydrogen−deuterium ex-change mass spectrometry (HDXMS), 33 and 13 C ENDOR spectroscopy, 34 has both enhanced our understanding of the structural origin of perturbed conformational landscapes and expanded the "deep-tunneling" model to include both local DAD sampling and more global conformational sampling coordinates. 9,35 For the majority of enzymes that have been studied (e.g., refs 32, 36−40), perturbations produce an elevated energy of activation for C−H cleavage that can be ascribed to an altered global conformational landscape with an increase in inactive (or poorly active) enzyme−substrate (E-S) substates.…”

Section: ■ Introductionmentioning

confidence: 99%

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Biophysical Characterization of a Disabled Double Mutant of Soybean Lipoxygenase: The “Undoing” of Precise Substrate Positioning Relative to Metal Cofactor and an Identified Dynamical Network

et al. 2019

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Soybean lipoxygenase (SLO) has served as a prototype for understanding the molecular origin of enzymatic rate accelerations. The double mutant (DM) L546A/L754A is considered a dramatic outlier, due to the unprecedented size and near temperature-independence of its primary kinetic isotope effect, low catalytic efficiency, and elevated enthalpy of activation. To uncover the physical basis of these features, we herein apply three structural probes: hydrogen-deuterium exchange mass spectrometry, room-temperature X-ray crystallography and EPR spectroscopy on four SLO variants (wild-type (WT) enzyme, DM, and the two parental single mutants, L546A and L754A). DM is found to incorporate features of each parent, with the perturbation at position 546 predominantly influencing thermally activated motions that connect the active site to a proteinsolvent interface, while mutation at position 754 disrupts the ligand field and solvation near the cofactor iron. However, the expanded active site in DM leads to more active site water molecules *

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Section: ■ Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Biophysical Characterization of a Disabled Double Mutant of Soybean Lipoxygenase: The “Undoing” of Precise Substrate Positioning Relative to Metal Cofactor and an Identified Dynamical Network

et al. 2019

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“…Numerically fitting these data then gives Δ H ‡ , Δ S ‡ , Δ G ‡ ,

Δ C_{P}^{‡}

, Δ V ‡ , Δβ ‡ and Δα ‡ reflecting the changes in enthalpy, entropy, Gibbs free energy, heat capacity, activation volume, compressibility and expansivity between the enzyme–substrate complex and the enzyme–transition state complex respectively. The enzyme kinetics for only a relatively few enzymes has been treated by combined p / T studies . In these cases, the p /T plane has not been fitted with a numerical model meaning at least

Δ C_{P}^{‡}

and ∆α ‡ are not determined.…”

Section: Introductionmentioning

confidence: 99%

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis

et al. 2017

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Our understanding of how enzymes work is coloured by static structure depictions where the enzyme scaffold is presented as either immobile, or in equilibrium between well-defined static conformations. Proteins, however, exhibit a large degree of motion over a broad range of timescales and magnitudes and this is defined thermodynamically by the enzyme free energy landscape (FEL). The role and importance of enzyme motion is extremely contentious. Much of the challenge is in the experimental detection of so called 'conformational sampling' involved in enzyme turnover. Herein we apply combined pressure and temperature kinetics studies to elucidate the full suite of thermodynamic parameters defining an enzyme FEL as it relates to enzyme turnover. We find that the key thermodynamic parameters governing vibrational modes related to enzyme turnover are the isobaric expansivity term and the change in heat capacity for enzyme catalysis. Variation in the enzyme FEL affects these terms. Our analysis is supported by a range of biophysical and computational approaches that specifically capture information on protein vibrational modes and the FEL (all atom flexibility calculations, red edge excitation shift spectroscopy and viscosity studies) that provide independent evidence for our findings. Our data suggest that restricting the enzyme FEL may be a powerful strategy when attempting to rationally engineer enzymes, particularly to alter thermal activity. Moreover, we demonstrate how rational predictions can be made with a rapid computational approach.

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“…Such dynamical interconversions are often rapid (picoseconds to microseconds) (14), and, due to their short lifetimes, exceedingly difficult to measure experimentally and to link to active site catalysis. Experimental tools to address these processes include the combined application of site-specific mutagenesis with biophysical measurements of fluorescence lifetimes and Stokes shifts (15), elevation of pressure (16), T-jump FRET (17), and rapid, time-dependent X-ray crystallography (18). Alternatively, hydrogen/deuterium exchange mass spectrometry (HDX-MS) can be used for the spatial resolution of protein motions (19)(20)(21)(22)(23)(24).…”

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confidence: 99%

Hydrogen deuterium exchange defines catalytically linked regions of protein flexibility in the catechol O -methyltransferase reaction

Zhang

Balsbaugh

Gao

et al. 2020

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

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Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor–acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954–7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15–20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.

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Hydrostatic Pressure Studies Distinguish Global from Local Protein Motions in C−H Activation by Soybean Lipoxygenase‐1

Cited by 5 publications

References 34 publications

Biophysical Characterization of a Disabled Double Mutant of Soybean Lipoxygenase: The “Undoing” of Precise Substrate Positioning Relative to Metal Cofactor and an Identified Dynamical Network

Biophysical Characterization of a Disabled Double Mutant of Soybean Lipoxygenase: The “Undoing” of Precise Substrate Positioning Relative to Metal Cofactor and an Identified Dynamical Network

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis

Hydrogen deuterium exchange defines catalytically linked regions of protein flexibility in the catechol O -methyltransferase reaction

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