Enzyme Dynamics along the Reaction Coordinate: Critical Role of a Conserved Residue

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The ability to use conformational flexibility is a hallmark of enzyme function. Here we show that protein motions and catalytic activity in a RNase are coupled and display identical solvent isotope effects. Solution NMR relaxation experiments identify a cluster of residues, some distant from the active site, that are integral to this motion. These studies implicate a single residue, histidine-48, as the key modulator in coupling protein motion with enzyme function. Mutation of H48 to alanine results in loss of protein motion in the isotope-sensitive region of the enzyme. In addition, kcat decreases for this mutant and the kinetic solvent isotope effect on kcat, which was 2.0 in WT, is near unity in H48A. Despite being located 18 Å from the enzyme active site, H48 is essential in coordinating the motions involved in the rate-limiting enzymatic step. These studies have identified, of Ϸ160 potential exchangeable protons, a single site that is integral in the rate-limiting step in RNase A enzyme function.Carr-Purcell-Meiboom-Gill dispersion ͉ enzyme dynamics ͉ NMR ͉ protein motions ͉ RNase A C onformational motions in enzymes play an essential role in their function and are often the rate-limiting step to overall catalytic throughput (1-5). Many enzymes have sufficiently evolved such that the bond-making and -breaking steps are fast relative to the ability of the enzyme to undergo a conformational change, and, thus, steps other than the chemical transformation of substrate are rate-limiting (ref. 6 and references therein). In systems such as these, understanding enzyme function requires characterization of the relevant time-dependent protein fluctuations from the timeaveraged three-dimensional structure. The ability of solution NMR spectroscopy to detect, with atomic resolution, motions over a wide timescale (picoseconds to seconds) makes it an ideal experimental technique to characterize conformational motions in proteins that can ultimately impact drug design, de novo enzyme construction, and enzyme engineering. In particular, relaxation-compensated Carr-Purcell-Meiboom-Gill (rcCPMG) dispersion experiments (7) are capable of informing on the kinetics, thermodynamics, and structural changes of protein motions in the microsecond-tomillisecond timescale.RNase A is an enzyme example in which a conformational change is the bottleneck to overall conversion of substrate to product (see ref. 3 for a review). RNase A catalyzes the cleavage of single-stranded RNA and does not require metal ions or cofactors. This enzyme has been studied in great detail as a model for protein folding, structure, and stability (8, 9). In addition, homologs of RNase A have important cytotoxic and antitumor properties (10). The rate-limiting step for the RNase A reaction is a protein conformational change that is coupled to the product release step (1, 11). This conformational change involves multiple amino acid residues throughout the protein, including those distant from the active site (12-14). These mobile regions include two loops: loop 1,...

Section: Resultsmentioning

confidence: 99%

Section: H48 Modulates Motion and Enzymementioning

confidence: 99%

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confidence: 99%

See 1 more Smart Citation

The mechanism of rate-limiting motions in enzyme function

Watt

Shimada

Kovrigin

et al. 2007

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145

201

“…Binding then occurs by a simple selection of those antigens whose epitopes are already in a matching conformation for the paratope. In general, growing support for conformational selection in specific protein-protein interactions is based mainly on finding bound-like conformations of proteins in the respective unbound ensembles of structures (12,14,(23)(24)(25)(26)(27)(28)(29)(30). For example, Gsponer et al (30) proposed that Ca 2ϩ -bound, ligand-free calmodulin samples the conformational space of calmodulin bound to myosin light chain kinase.…”

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confidence: 99%

Conformational selection and induced fit mechanism underlie specificity in noncovalent interactions with ubiquitin

Włodarski

Žagrović

2009

180

215

Noncovalent binding interactions between proteins are the central physicochemical phenomenon underlying biological signaling and functional control on the molecular level. Here, we perform an extensive structural analysis of a large set of bound and unbound ubiquitin conformers and study the level of residual induced fit after conformational selection in the binding process. We show that the region surrounding the binding site in ubiquitin undergoes conformational changes that are significantly more pronounced compared with the whole molecule on average. We demonstrate that these induced-fit structural adjustments are comparable in magnitude to conformational selection. Our final model of ubiquitin binding blends conformational selection with the subsequent induced fit and provides a quantitative measure of their respective contributions.ubiquitin binding ͉ protein recognition ͉ Kolmogorov-Smirnov test T he picture of protein-protein interactions has, over the decades, evolved from the early lock-and-key hypothesis (1) to the generally accepted and widely applied induced-fit model (2, 3). However, several different systems have recently been shown to follow an alternative paradigm whose central element is the idea of conformational selection (4). Within this paradigm, the conformational change in binding is thought to originate primarily from the conformational diversity of the unbound state (5-15). Simply put, the unbound protein explores the energy landscape, spending most of the time in the lowest energy conformations, but also occupying higher-energy ones, some of which are structurally similar to the bound conformations. In the course of binding, because of favorable interactions with the ligand, these conformers get preferentially selected and the population of protein microstates shifts in the direction of bound conformations (4-15). In a way, induced fit and conformational selection are two extremes of possible mechanisms underlying protein interactions (16): in the former, optimal binding is achieved by specific structural change, whereas in the latter it is brought about through selection from the already present unbound ensemble. The two mechanisms have recently been compared from the perspective of kinetics (17) and the energy landscape theory (18).Some of the earliest-described examples of the conformational selection paradigm are the antibody-antigen interactions where an antibody can be found in different unbound conformations, exhibiting different specificity for different antigens (19)(20)(21)(22). Binding then occurs by a simple selection of those antigens whose epitopes are already in a matching conformation for the paratope. In general, growing support for conformational selection in specific protein-protein interactions is based mainly on finding bound-like conformations of proteins in the respective unbound ensembles of structures (12,14,(23)(24)(25)(26)(27)(28)(29)(30). For example, Gsponer et al. (30) proposed that Ca 2ϩ -bound, ligand-free calmodulin samples the conformational space of ...

“…1, K 3 ) binding to the active state conformation shifts the population of free molecules toward the active state, facilitating further substrate binding. NMR studies of enzymes such as RNase A (16,17), cyclophilin A (18), and dihydrofolate reductase (19) provide evidence for conformational selection because the experimental data supports that even in the absence of ligand the enzyme samples multiple conformational states including the ligand-bound (active) state.…”

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confidence: 99%

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

Sullivan

Holyoak

2008

147

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 ...