Dynamics of a flexible loop in dihydrofolate reductase from Escherichia coli and its implication for catalysis

126

262

Protein dynamics have controversially been proposed to be at the heart of enzyme catalysis, but identification and analysis of dynamical effects in enzyme-catalyzed reactions have proved very challenging. Here, we tackle this question by comparing an enzyme with its heavy ( 15 N, 13 C, 2 H substituted) counterpart, providing a subtle probe of dynamics. The crucial hydride transfer step of the reaction (the chemical step) occurs more slowly in the heavy enzyme. A combination of experimental results, quantum mechanics/molecular mechanics simulations, and theoretical analyses identify the origins of the observed differences in reactivity. The generally slightly slower reaction in the heavy enzyme reflects differences in environmental coupling to the hydride transfer step. Importantly, the barrier and contribution of quantum tunneling are not affected, indicating no significant role for "promoting motions" in driving tunneling or modulating the barrier. The chemical step is slower in the heavy enzyme because protein motions coupled to the reaction coordinate are slower. The fact that the heavy enzyme is only slightly less active than its light counterpart shows that protein dynamics have a small, but measurable, effect on the chemical reaction rate.kinetics | computational chemistry | biological chemistry | biophysics | quantum biology

Section: Resultssupporting

confidence: 85%

Unraveling the role of protein dynamics in dihydrofolate reductase catalysis

Luk

Ruiz‐Pernía

Dawson

et al. 2013

126

262

“…It has been demonstrated previously in several enzyme systems that the motion of active site loop regions is either rate limiting or intimately coupled to the chemical step (39)(40)(41)(42). Previous structural studies have demonstrated that the predominant state for the active site lid domain in most complexes of PEPCK studied to date is the open/disordered state (28,29,32,35).…”

Section: Induced Fit and Conformational Selectionmentioning

confidence: 99%

Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection

Sullivan

Holyoak

2008

143

217

The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics and catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.enzyme dynamics ͉ population shift ͉ phosphoenolpyruvate carboxykinase I t has been 50 years since the original presentation of the induced fit hypothesis by Daniel Koshland (1) that built upon Emil Fisher's key-lock principle (2) and introduced the idea that the inherent dynamic properties of enzymes play an essential role in the processes of molecular recognition and catalysis. Over the last decade, with the advent of new instrumentation and novel experimental approaches there has been a resurgence in investigations focused upon understanding the specific ways in that these dynamic properties influence the ability of enzymes to achieve enormous levels of selectivity and catalytic power (refs. 3-6 and references therein).Presently there are two primary models used to explain the mechanism by which an enzyme can move toward adopting the final key-lock state ( Fig. 1): (i) the induced fit model and (ii) the conformational selection/population shift model (7-14). As defined originally by Koshland (1), the induced fit model states that the achievement of the final key-lock state, that is, the precise orientation of catalytic groups and residues necessary for catalysis, occurs only after changes in protein structure that are induced by the binding of a substrate. This pathway is represented by the equilibrium constants K 1 and K 2 in Fig. 1. Intrinsic to this model is the ability of the enzyme to form an ''encounter complex'' † (Fig. 1B). In this complex the enzyme is liganded identically to the eventual catalytically competent key-lock state; however, the conformational change leading to the active key-lock state has not yet occurred. In contrast, the conformational selection model states that the enzyme exists in multiple conformational states, of which the substrate has a high affinity for the active or key-lock state. In this model the ligand is suggested to bind to this lowly populated high-energy ...

“…Protein dynamics on the ps-ns and μs-ms timescales have been examined for a number of DHFR complexes by using NMR-based techniques (12)(13)(14)(15)(16)(17). The E:FOL and E:FOL: 5′,6′-dihydroNADPH (DHNADPH) complexes are both in the occluded conformation and display very similar ps-ns backbone dynamics that differ from those of the closed E:FOL:NADP þ complex (16).…”

mentioning

confidence: 99%

Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands

Boehr

McElheny

Dyson

et al. 2010

Self Cite

130

171

Enzyme catalysis can be described as progress over a multidimensional energy landscape where ensembles of interconverting conformational substates channel the enzyme through its catalytic cycle. We applied NMR relaxation dispersion to investigate the role of bound ligands in modulating the dynamics and energy landscape of Escherichia coli dihydrofolate reductase to obtain insights into the mechanism by which the enzyme efficiently samples functional conformations as it traverses its reaction pathway. Although the structural differences between the occluded substrate binary complexes and product ternary complexes are very small, there are substantial differences in protein dynamics. Backbone fluctuations on the μs-ms timescale in the cofactor binding cleft are similar for the substrate and product binary complexes, but fluctuations on this timescale in the active site loops are observed only for complexes with substrate or substrate analog and are not observed for the binary product complex. The dynamics in the substrate and product binary complexes are governed by quite different kinetic and thermodynamic parameters. Analogous dynamic differences in the E:THF:NADPH and E:THF:NADP þ product ternary complexes are difficult to rationalize from ground-state structures. For both of these complexes, the nicotinamide ring resides outside the active site pocket in the ground state. However, they differ in the structure, energetics, and dynamics of accessible higher energy substates where the nicotinamide ring transiently occupies the active site. Overall, our results suggest that dynamics in dihydrofolate reductase are exquisitely "tuned" for every intermediate in the catalytic cycle; structural fluctuations efficiently channel the enzyme through functionally relevant conformational space. catalysis | energy landscape | enzyme dynamics | NMR | relaxation