Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Protein motions and the activation of the CH bond catalyzed by dihydrofolate reductase.

ABSTRACT: The role of protein motions in enzymatic CH?C transfer is an area of great contemporary debate. An effective tool in probing such a role is the temperature dependence of the intrinsic kinetic isotope effects for the enzyme-catalyzed reaction. The outcome of those experiments is interpreted within the context of phenomenological Marcus-like models of hydrogen tunneling. The current review focuses on recent studies of dihydrofolate reductase (DHFR) and how the role of protein motions in the catalyzed reaction has been demonstrated. The motions in DHFR are controlled by local effects of active site residues, global effects involving remote residues across the enzyme and appear to be preserved during the evolution of the enzyme from bacteria to human.

SUBMITTER: Francis K

PROVIDER: S-EPMC4149937 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Protein motions and the activation of the CH bond catalyzed by dihydrofolate reductase.

Francis Kevin K Kohen Amnon A

Current opinion in chemical biology 20140416

The role of protein motions in enzymatic CH→C transfer is an area of great contemporary debate. An effective tool in probing such a role is the temperature dependence of the intrinsic kinetic isotope effects for the enzyme-catalyzed reaction. The outcome of those experiments is interpreted within the context of phenomenological Marcus-like models of hydrogen tunneling. The current review focuses on recent studies of dihydrofolate reductase (DHFR) and how the role of protein motions in the cataly ...[more]

PMID: 24742825

Similar Datasets

Temporally overlapped but uncoupled motions in dihydrofolate reductase catalysis.

Project description:Temporal correlations between protein motions and enzymatic reactions are often interpreted as evidence for catalytically important motions. Using dihydrofolate reductase as a model system, we show that there are many protein motions that temporally overlapped with the chemical reaction, and yet they do not exhibit the same kinetic behaviors (KIE and pH dependence) as the catalyzed chemical reaction. Thus, despite the temporal correlation, these motions are not directly coupled to the chemical transformation, and they might represent a different part of the catalytic cycle or simply be the product of the intrinsic flexibility of the protein.

| S-EPMC3779903 | biostudies-literature

Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase.

Project description:The enzyme DHFR (dihydrofolate reductase) catalyses hydride transfer from NADPH to, and protonation of, dihydrofolate. The physical basis of the hydride transfer step catalysed by DHFR from Escherichia coli has been studied through the measurement of the temperature dependence of the reaction rates and the kinetic isotope effects. Single turnover experiments at pH 7.0 revealed a strong dependence of the reaction rates on temperature. The observed relatively large difference in the activation energies for hydrogen and deuterium transfer led to a temperature dependence of the primary kinetic isotope effects from 3.0+/-0.2 at 5 degrees C to 2.2+/-0.2 at 40 degrees C and an inverse ratio of the pre-exponential factors of 0.108+/-0.04. These results are consistent with theoretical models for hydrogen transfer that include contributions from quantum mechanical tunnelling coupled with protein motions that actively modulate the tunnelling distance. Previous work had suggested a coupling of a remote residue,Gly121, with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121V-DHFR, in which Gly121 was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G121V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121V-DHFR catalysis. In contrast, steady-state measurements at pH 9.5, where hydride transfer is rate limiting, revealed temperature-independent kinetic isotope effects between 15 and 35 degrees C and a ratio of the pre-exponential factors above the semi-classical limit, suggesting a rigid active site configuration from which hydrogen tunnelling occurs. The mechanism by which hydrogen tunnelling in DHFR is coupled with the environment appears therefore to be sensitive to pH.

| S-EPMC1386024 | biostudies-literature

The role of large-scale motions in catalysis by dihydrofolate reductase.

Project description:Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR.

| S-EPMC3590880 | biostudies-literature

Temperature dependence of protein motions in a thermophilic dihydrofolate reductase and its relationship to catalytic efficiency.

Project description:We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability, probed with circular dichroism, established an accessible temperature range of 10 degrees C to 55 degrees C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, DeltaH degrees (unf(avg)). Most regions in the enzyme show DeltaH degrees (unf(avg)) < or = 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for DeltaH degrees (unf(avg)) are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate.

| S-EPMC2890430 | biostudies-literature

Site-Specific Tryptophan Labels Reveal Local Microsecond-Millisecond Motions of Dihydrofolate Reductase.

Project description:Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood. Escherichia coli dihydrofolate reductase (DHFR) is a small, flexible enzyme that is often used as a model system for understanding enzyme dynamics. Recently, native tryptophan fluorescence was used as a probe to study micro- to millisecond dynamics of DHFR. Yet, because DHFR has five native tryptophans, the origin of the observed conformational changes could not be assigned to a specific region within the enzyme. Here, we use DHFR mutants, each with a single tryptophan as a probe for temperature jump fluorescence spectroscopy, to further inform our understanding of DHFR dynamics. The equilibrium tryptophan fluorescence of the mutants shows that each tryptophan is in a different environment and that wild-type DHFR fluorescence is not a simple summation of all the individual tryptophan fluorescence signatures due to tryptophan-tryptophan interactions. Additionally, each mutant exhibits a two-phase relaxation profile corresponding to ligand association/dissociation convolved with associated conformational changes and a slow conformational change that is independent of ligand association and dissociation, similar to the wild-type enzyme. However, the relaxation rate of the slow phase depends on the location of the tryptophan within the enzyme, supporting the conclusion that the individual tryptophan fluorescence dynamics do not originate from a single collective motion, but instead report on local motions throughout the enzyme.

| S-EPMC7503464 | biostudies-literature

Catalysis by dihydrofolate reductase and other enzymes arises from electrostatic preorganization, not conformational motions.

Project description:The proposal that enzymatic catalysis is due to conformational fluctuations has been previously promoted by means of indirect considerations. However, recent works have focused on cases where the relevant motions have components toward distinct conformational regions, whose population could be manipulated by mutations. In particular, a recent work has claimed to provide direct experimental evidence for a dynamical contribution to catalysis in dihydrofolate reductase, where blocking a relevant conformational coordinate was related to the suppression of the motion toward the occluded conformation. The present work utilizes computer simulations to elucidate the true molecular basis for the experimentally observed effect. We start by reproducing the trend in the measured change in catalysis upon mutations (which was assumed to arise as a result of a "dynamical knockout" caused by the mutations). This analysis is performed by calculating the change in the corresponding activation barriers without the need to invoke dynamical effects. We then generate the catalytic landscape of the enzyme and demonstrate that motions in the conformational space do not help drive catalysis. We also discuss the role of flexibility and conformational dynamics in catalysis, once again demonstrating that their role is negligible and that the largest contribution to catalysis arises from electrostatic preorganization. Finally, we point out that the changes in the reaction potential surface modify the reorganization free energy (which includes entropic effects), and such changes in the surface also alter the corresponding motion. However, this motion is never the reason for catalysis, but rather simply a reflection of the shape of the reaction potential surface.

| S-EPMC3161549 | biostudies-literature

Lessons from nature: unraveling biological CH bond activation.

Project description:The cleavage of unactivated CH bonds is one of the most challenging reactions in chemical biology. Metalloenzymes have evolved that efficiently perform these transformations with exquisite control of selectivity; however, a proposed requirement is the generation of highly reactive intermediates that could be lethal. A thermodynamic argument involving the putative reactive species is outlined, whereby the interplay between two tunable parameters, redox potential and pK(a), may be the key to sustainable function. In addition, factors that control these parameters are also described, including hydrogen-bonding networks found within protein active sites. Synthetic examples are used to corroborate these ideas.

| S-EPMC2676355 | biostudies-literature

Extension and limits of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase.

Project description:Enzyme catalysis has been studied extensively, but the role of enzyme dynamics in the catalyzed chemical conversion is still an enigma. The enzyme dihydrofolate reductase (DHFR) is often used as a model system to assess a network of coupled motions across the protein that may affect the catalyzed chemical transformation. Molecular dynamics simulations, quantum mechanical/molecular mechanical studies, and bioinformatics studies have suggested the presence of a "global dynamic network" of residues in DHFR. Earlier studies of two DHFR distal mutants, G121V and M42W, indicated that these residues affect the chemical step synergistically. While this finding was in accordance with the concept of a network of functional motions across the protein, two residues do not constitute a network. To better define the extent and limits of the proposed network, the current work studied two remote residues predicted to be part of the same network: W133 and F125. The effect of mutations in these residues on the nature of the chemical step was examined via measurements of the temperature-dependence of the intrinsic kinetic isotope effects (KIEs) and other kinetic parameters, and double mutants were used to tie the findings to G121 and M42. The findings indicate that residue F125, which was implicated by both calculations and bioinformatic methods, is a part of the same global dynamic network as G121 and M42, while W133, implicated only by bioinformatics, is not. These findings extend our understanding of the proposed network and the relations between functional and genomic couplings. Delineating that network illuminates the need to consider remote residues and protein structural dynamics in the rational design of drugs and of biomimetic catalysts.

| S-EPMC3985941 | biostudies-literature

Hydride transfer catalysed by Escherichia coli and Bacillus subtilis dihydrofolate reductase: coupled motions and distal mutations.

Project description:This paper reviews the results from hybrid quantum/classical molecular dynamics simulations of the hydride transfer reaction catalysed by wild-type (WT) and mutant Escherichia coli and WT Bacillus subtilis dihydrofolate reductase (DHFR). Nuclear quantum effects such as zero point energy and hydrogen tunnelling are significant in these reactions and substantially decrease the free energy barrier. The donor-acceptor distance decreases to ca 2.7 A at transition-state configurations to enable the hydride transfer. A network of coupled motions representing conformational changes along the collective reaction coordinate facilitates the hydride transfer reaction by decreasing the donor-acceptor distance and providing a favourable geometric and electrostatic environment. Recent single-molecule experiments confirm that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time-scale of the hydride transfer. Distal mutations can lead to non-local structural changes and significantly impact the probability of sampling configurations conducive to the hydride transfer, thereby altering the free-energy barrier and the rate of hydride transfer. E. coli and B. subtilis DHFR enzymes, which have similar tertiary structures and hydride transfer rates with 44% sequence identity, exhibit both similarities and differences in the equilibrium motions and conformational changes correlated to hydride transfer, suggesting a balance of conservation and flexibility across species.

| S-EPMC1647314 | biostudies-literature

Preservation of protein dynamics in dihydrofolate reductase evolution.

Project description:The hydride transfer reaction catalyzed by dihydrofolate reductase (DHFR) is a model for examining how protein dynamics contribute to enzymatic function. The relationship between functional motions and enzyme evolution has attracted significant attention. Recent studies on N23PP Escherichia coli DHFR (ecDHFR) mutant, designed to resemble parts of the human enzyme, indicated a reduced single turnover rate. NMR relaxation dispersion experiments with that enzyme showed rigidification of millisecond Met-20 loop motions (Bhabha, G., Lee, J., Ekiert, D. C., Gam, J., Wilson, I. A., Dyson, H. J., Benkovic, S. J., and Wright, P. E. (2011) Science 332, 234-238). A more recent study of this mutant, however, indicated that fast motions along the reaction coordinate are actually more dispersed than for wild-type ecDHFR (WT). Furthermore, a double mutant (N23PP/G51PEKN) that better mimics the human enzyme seems to restore both the single turnover rates and narrow distribution of fast dynamics (Liu, C. T., Hanoian, P., French, T. H., Hammes-Schiffer, S., and Benkovic, S. J. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 10159-11064). Here, we measured intrinsic kinetic isotope effects for both N23PP and N23PP/G51PEKN double mutant DHFRs over a temperature range. The findings indicate that although the C-H?C transfer and dynamics along the reaction coordinate are impaired in the altered N23PP mutant, both seem to be restored in the N23PP/G51PEKN double mutant. This indicates that the evolution of G51PEKN, although remote from the Met-20 loop, alleviated the loop rigidification that would have been caused by N23PP, enabling WT-like H-tunneling. The correlation between the calculated dynamics, the nature of C-H?C transfer, and a phylogenetic analysis of DHFR sequences are consistent with evolutionary preservation of the protein dynamics to enable H-tunneling from well reorganized active sites.

| S-EPMC3861645 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data