Importance of protein dynamics during enzymatic C-H bond cleavage catalysis

Abstract

Quantum tunneling and protein dynamics have emerged as important components of enzyme function. This review focuses on soybean lipoxygenase-1, to illustrate how the properties of enzymatic C-H bond activation link protein motions to the fundamental bond making-breaking processes.

Figure 1

Illustrative free energy profiles and Arrhenius plots for the cleavage of a C–L bond, where L is H, D, or T. (A) Semiclassical KIEs predict extrapolation at high temperatures to Arrhenius prefactors that are close to being independent of isotopic labeling. (B) Tunneling correction models predict a larger value for both E_a and A_L in the case of the heavier isotope(s). (C) Full tunneling models are able to reproduce data where there is no or little difference in the magnitude of E_a among the isotopes and values of A_L that are greatly elevated for H in relation to D and T.

Figure 2

Rate constant for tunneling, as formulated in ref , comprised of three exponential terms. (a) Heavy atom motions that lead to degeneracy of reactant and product wells, a prerequisite for hydrogen tunneling between the donor and acceptor. The rate of reaching this state depends on the reaction driving force (ΔG°) and the reorganization energy (λ). (b) Resulting wave function overlap that is dependent on mass (m_H), frequency (ω_H), and distance (r_H). The wave function overlap is shown as being greater for protium than for deuterium. (c) Energy dependence (E_x) for sampling of different donor–acceptor distances (r_x). The increased energy at shorter donor–acceptor distances is offset by a more efficient wave function overlap. Adapted from ref .

Figure 3

Model to illustrate the impact of active site mutations in ht-ADH above and below the transition temperature at 30 °C. State 1 is the ideal state and represents the WT protein above 30 °C. The protein is optimally flexible (in the black region), and this generates active site compression that is accompanied by a close approach of the H-donor and H-acceptor. State 2 represents the situation following active site mutation above 30 °C. The white region, representing the active site, is shown in an artificially enlarged manner to allow a depiction of the resulting increased distance between the H-donor and H-acceptor that is accompanied by a decrease in the force constant for DAD sampling. State 3 is the situation that results from the combination of active site mutation and low temperature. Once again, the region representing the active site (white) is enlarged, to allow depiction of the increase in the donor–acceptor distance. Under the conditions of state 3, the increased rigidity of the protein below 30 °C prevents any “recovery” via DAD sampling and the KIE once again becomes temperature-independent.

Figure 4

Dependence of the chemical coordinate (Reaction Coordinate) on the conformational substates (Ensemble Conformations). As illustrated, each conformational substate converts substrate to product with a distinctive rate constant (provided by R. Larsen and A. Kohen). A similar picture has been introduced by Benkovic et al. to illustrate the role of a conformational landscape in enzyme catalysis.

Figure 5

X-ray structure of SLO-1, with LA modeled into the active site.

Figure 6

Schematic impact of deleterious mutation on the conformational landscape of SLO-1. The conformational landscape of the enzyme is shown by solid black lines with energy increasing along the vertical axis. Blue indicates active conformers and red inactive conformer(s). In the case of WT SLO-1, the enzyme molecules are fully populated in the catalytically active regions of the conformational spaces. In the double mutant, Leu546Ala/Leu754Ala, only a small proportion of the enzyme is populated in the catalytically active region of the conformational space and a significant proportion of enzyme occupies deep local minima of catalytically very slow and/or inactive conformations. For the sake of simplicity, only one deep local minimum is shown. Different conformational landscapes are expected for the free enzyme (controlling k_cat/K_m(LA)) and the E·S′ complex (controlling k_cat). In the example shown, the fraction of active enzyme is obtained from the relative k_cat numbers.

Scheme 1. Reaction Mechanism for Soybean Lipoxygenase-1

Scheme 2. Diagram To Illustrate the Expected Consequences for k_cat/K_m(LA) in the Event of a Decrease in the Rate Constant To Reach the Reactive Enzyme–Substrate Complex (E–S) via TS-1 (dashed line) versus a Decrease in the Rate Constant for C–H Bond Cleavage via TS-2 (dashed red line)^a

^aThe standard state for the binding of substrate has been arbitrarily set at the K_d for substrate, resulting in equal energies for E+S and E·S.

Scheme 3. Summary of Experimental and Computational Secondary Deuterium KIEs at Positions 9, 10, 12, and 13 of LA.^a

^a(A) Reaction of [11-H₂]LA, corrected for kinetic complexity. (B) Reaction of [11-²H₂]LA. (C) Computed KIEs using an optimized structure for LA bound to SLO-1 and the assumption of a freely equilibrated pentadienyl radical intermediate (cf. Scheme 1).