Evolution of the Chemical Step in Enzyme Catalysis

Singh, Priyanka; Vandemeulebroucke, An; Li, Jiayue; Schulenburg, Cindy; Fortunato, Gabriel; Kohen, Amnon; Hilvert, Donald; Cheatum, Christopher M.

doi:10.1021/acscatal.1c00442

Cited by 21 publications

(27 citation statements)

References 48 publications

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“…An increase in ΔE a has been shown to map to a loss of precision in the local positioning of the reactive carbon (C11 of the LA substrate) for SLO (53), as well as for a wide range of H-transfer enzymes (54). In accordance with this behavior, evolutionary selection was recently applied to a primitive form of dihydrofolate reductase (DHFR), showing a progressive decrease in ΔE a as the evolved enzyme approached the activity of native DHFRs (55). From this study of I552A and V750A, we conclude that ΔE a is largely unaffected, with the impact of such mutations appearing primarily as a reduction in the enthalpy of activation (E a (H)) that is accompanied by reduction of the first-order rate constant ( k cat ) at 30 °C.…”

Section: Resultsmentioning

confidence: 99%

Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

Zaragoza

Offenbacher

Shi-sheng

et al. 2022

Preprint

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The enzyme soybean lipoxygenase provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen deuterium exchange experiments to detect a radiating cone of aliphatic side chains that extends from the iron active site of SLO to the protein-solvent interface. Employing eight variants of SLO, nanosecond fluorescence Stokes shifts have been measured using a probe appended to the identified surface loop. We report a remarkable identity of the enthalpies of activation for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within the identified thermal network. While the role of dynamics in enzyme function has been predominantly attributed to a rapidly equilibrating protein conformational landscape, these new data implicate a rapid and cooperative protein quake as the origin of the thermal activation of SLO. A mechanism of catalysis is presented that combines a distributed conformational landscape with a long range and site-specific thermal quake.

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Section: Resultsmentioning

confidence: 99%

Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

Zaragoza

Offenbacher

Shi-sheng

et al. 2022

Preprint

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“…Six kinetic runs of 12 half-lives of the reaction were measured for each isotopic reaction back to back. The procedure was then repeated at other temperatures as quickly as possible (5,15,25,35,45 °C, in order) so that the instrument settings were kept the same and the aging of the reaction solutions was the minimum. (The solutions were wrapped with aluminum foil and kept in a refrigerator between temperatures.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Solvent Effects on the Temperature Dependence of Hydride Kinetic Isotope Effects: Correlation to the Donor–Acceptor Distances

et al. 2022

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Protein structural effects on the temperature (T) dependence of kinetic isotope effects (KIEs) in H-tunneling reactions have recently been used to discuss about the role of enzyme thermal motions in catalysis. Frequently observed nearly T-independent KIEs in the wild-type enzymes and T-dependent KIEs in variants suggest that H-tunneling in the former is assisted by the naturally evolved protein constructive vibrations that help sample short donor–acceptor distances (DADs) needed. This explanation that correlates the T-dependence of KIEs with DAD sampling has been highly debated as simulations following other H-tunneling models sometimes gave alternative explanations. In this paper, solvent effects on the T-dependence of KIEs of two hydride tunneling reactions of NADH/NAD+ analogues (represented by ΔE a = E aD – E aH) were determined in attempts to replicate the observations in enzymes and test the protein vibration-assisted DAD sampling concept. Effects of selected aprotic solvents on the DADPRC’s of the productive reactant complexes (PRCs) and the DADTRS’s of the activated tunneling ready states (TRSs) were obtained through computations and analyses of the kinetic data, including 2° KIEs, respectively. A weaker T-dependence of KIEs (i.e., smaller ΔE a) was found in a more polar aprotic solvent in which the system has a shorter average DADPRC and DADTRS. Further results show that a charge-transfer (CT) complexation made of a stronger donor/acceptor gives rise to a smaller ΔE a. Overall, the shorter and less broadly distributed DADs resulting from the stronger CT complexation vibrations give rise to a smaller ΔE a. Our results appear to support the explanation that links the T-dependence of KIEs to the donor–acceptor rigidity in enzymes.

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“…Conformational selection enabled by protein flexibility is fundamental to enzyme catalysis. − Directed changes in protein conformation (“coupled motions”) enable enzymes to coordinate the immediate catalytic event with other physical steps of the reaction, such as substrate binding and product release. − In the chemical transformation on the enzyme, stochastic motions enable the dynamical population (“sampling”) of catalytically relevant ground-state conformers to have electrostatics and internuclear distances tuned for bond cleavage/formation. − On this dynamic view, conformational selection connects directly to catalytic rate enhancement; and protein flexibility represents an evolutionary target for the optimization of enzyme efficiency. − Enzymes of the alcohol dehydrogenase class (ADHs) ,− as well as several other oxidoreductases (e.g., dihydrofolate reductase, ,,− thymidylate synthase, , formate dehydrogenase, , flavin-dependent ene-reductases, − lipoxygenases , ) have been instrumental to link conformational selection to catalysis. ADHs promote hydride transfer between the substrate and nicotinamide coenzyme. ,,, Protein flexibility enables ADHs to sample conformers that place the hydride donor in close proximity to the acceptor. ,,− Reaction occurs classically over the enthalpic barrier but also by quantum mechanical tunneling. ,− Conformational selection of donor–acceptor distances (DADs) suitable for tunneling emphasizes dynamic control of the barrier width, in addition to a decrease in the barri...…”

Section: Introductionmentioning

confidence: 99%

Hydride Transfer Mechanism of Enzymatic Sugar Nucleotide C2 Epimerization Probed with a Loose-Fit CDP-Glucose Substrate

Rapp

Nidetzky

2022

ACS Catal.

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Transient oxidation–reduction through hydride transfer with tightly bound NAD coenzyme is used by a large class of sugar nucleotide epimerases to promote configurational inversion of carbon stereocenters in carbohydrate substrates. A requirement for the epimerases to coordinate hydride abstraction and re-addition with substrate rotation in the binding pocket poses a challenge for dynamical protein conformational selection linked to enzyme catalysis. Here, we studied the thermophilic C2 epimerase from Thermodesulfatator atlanticus ( Ta CPa2E) in combination with a slow CDP-glucose substrate ( k cat ≈ 1.0 min –1 ; 60 °C) to explore the sensitivity of the enzymatic hydride transfer toward environmental fluctuations affected by temperature (20–80 °C). We determined noncompetitive primary kinetic isotope effects (KIE) due to 2 H at the glucose C2 and showed that a normal KIE on the k cat ( D k cat ) reflects isotope sensitivity of the hydrogen abstraction to enzyme-NAD + in a rate-limiting transient oxidation. The D k cat peaked at 40 °C was 6.1 and decreased to 2.1 at low (20 °C) and 3.3 at high temperature (80 °C). The temperature profiles for k cat with the 1 H and 2 H substrate showed a decrease in the rate below a dynamically important breakpoint (∼40 °C), suggesting an equilibrium shift to an impaired conformational landscape relevant for catalysis in the low-temperature region. Full Marcus-like model fits of the rate and KIE profiles provided evidence for a high-temperature reaction via low-frequency conformational sampling associated with a broad distribution of hydride donor–acceptor distances (long-distance population centered at 3.31 ± 0.02 Å), only poorly suitable for quantum mechanical tunneling. Collectively, dynamical characteristics of Ta CPa2E-catalyzed hydride transfer during transient oxidation of CDP-glucose reveal important analogies to mechanistically simpler enzymes such as alcohol dehydrogenase and dihydrofolate reductase. A loose-fit substrate (in Ta CPa2E) resembles structural variants of these enzymes by extensive dynamical sampling to balance conformational flexibility and catalytic efficiency.

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Evolution of the Chemical Step in Enzyme Catalysis

Cited by 21 publications

References 48 publications

Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

Solvent Effects on the Temperature Dependence of Hydride Kinetic Isotope Effects: Correlation to the Donor–Acceptor Distances

Hydride Transfer Mechanism of Enzymatic Sugar Nucleotide C2 Epimerization Probed with a Loose-Fit CDP-Glucose Substrate

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