Browse
Submit Data
Databases
API
Help

Dataset Information

28 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

ABSTRACT: States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

SUBMITTER: Zeymer C

PROVIDER: S-EPMC5026167 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

Zeymer Cathleen C Werbeck Nicolas D ND Zimmermann Sabine S Reinstein Jochen J Hansen D Flemming DF

Angewandte Chemie (International ed. in English) 20160818 38

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential argin ...[more]

PMID: 27534930

Similar Datasets

Energy landscape along an enzymatic reaction trajectory: hinges or cracks?

Project description:Are the most dynamically flexible regions around the equilibrium structure of an enzyme the same regions involved in the transition state for rate limiting processes involved in the enzymatic reaction? Kern and-coworkers (Wolf-Watzet al., 2004; Henzler-Wildman et al., 2007a, 2007b) have shown that insights about functionally relevant motions that determine the overall enzyme turnover rate can be obtained by investigating conformational dynamics around the equilibrium basin of the enzyme adenylate kinase. An allosteric change in protein structure turns out to be the controlling process. In this commentary we compare results of this study with earlier predictions of the route by which the enzyme undergoes its conformational change. These predictions are based on the idea that the energy surface for the protein is determined by the end structures of the conformational change. A key issue is whether the protein moves by specific hinges or whether it "cracks" and accesses partially unfolded states during its structural change.

| S-EPMC2645576 | biostudies-other

Shift in Conformational Equilibrium Underlies the Oscillatory Phosphoryl Transfer Reaction in the Circadian Clock.

Project description:Oscillatory phosphorylation/dephosphorylation can be commonly found in a biological system as a means of signal transduction though its pivotal presence in the workings of circadian clocks has drawn significant interest: for example in a significant portion of the physiology of Synechococcus elongatus PCC 7942. The biological oscillatory reaction in the cyanobacterial circadian clock can be visualized through its reconstitution in a test tube by mixing three proteins-KaiA, KaiB and KaiC-with adenosine triphosphate and magnesium ions. Surprisingly, the oscillatory phosphorylation/dephosphorylation of the hexameric KaiC takes place spontaneously and almost indefinitely in a test tube as long as ATP is present. This autonomous post-translational modification is tightly regulated by the conformational change of the C-terminal peptide of KaiC called the "A-loop" between the exposed and the buried states, a process induced by the time-course binding events of KaiA and KaiB to KaiC. There are three putative hydrogen-bond forming residues of the A-loop that are important for stabilizing its buried conformation. Substituting the residues with alanine enabled us to observe KaiB's role in dephosphorylating hyperphosphorylated KaiC, independent of KaiA's effect. We found a novel role of KaiB that its binding to KaiC induces the A-loop toward its buried conformation, which in turn activates the autodephosphorylation of KaiC. In addition to its traditional role of sequestering KaiA, KaiB's binding contributes to the robustness of cyclic KaiC phosphorylation by inhibiting it during the dephosphorylation phase, effectively shifting the equilibrium toward the correct phase of the clock.

| S-EPMC8538168 | biostudies-literature

Flexibility and Design: Conformational Heterogeneity along the Evolutionary Trajectory of a Redesigned Ubiquitin.

Project description:Although protein design has been used to introduce new functions, designed variants generally only function as well as natural proteins after rounds of laboratory evolution. One possibility for this pattern is that designed mutants frequently sample nonfunctional conformations. To test this idea, we exploited advances in multiconformer modeling of room-temperature X-ray data collection on redesigned ubiquitin variants selected for increasing binding affinity to the deubiquitinase USP7. Initial core mutations disrupt natural packing and lead to increased flexibility. Additional, experimentally selected mutations quenched conformational heterogeneity through new stabilizing interactions. Stabilizing interactions, such as cation-pi stacking and ordered waters, which are not included in standard protein design energy functions, can create specific interactions that have long-range effects on flexibility across the protein. Our results suggest that increasing flexibility may be a useful strategy to escape local minima during initial directed evolution and protein design steps when creating new functions.

| S-EPMC5415430 | biostudies-literature

Conformational motions regulate phosphoryl transfer in related protein tyrosine phosphatases.

Project description:Many studies have implicated a role for conformational motions during the catalytic cycle, acting to optimize the binding pocket or facilitate product release, but a more intimate role in the chemical reaction has not been described. We address this by monitoring active-site loop motion in two protein tyrosine phosphatases (PTPs) using nuclear magnetic resonance spectroscopy. The PTPs, YopH and PTP1B, have very different catalytic rates; however, we find in both that the active-site loop closes to its catalytically competent position at rates that mirror the phosphotyrosine cleavage kinetics. This loop contains the catalytic acid, suggesting that loop closure occurs concomitantly with the protonation of the leaving group tyrosine and explains the different kinetics of two otherwise chemically and mechanistically indistinguishable enzymes.

| S-EPMC4078984 | biostudies-literature

Damped Oscillating Phosphoryl Transfer Reaction in the Cyanobacterial Circadian Clock.

Project description:Most organisms have circadian clocks to ensure the metabolic cycle to resonate with the rhythmic environmental changes without "damping," or losing robustness. Cyanobacteria is the oldest and simplest form of life that is known to harbor this biological intricacy. Its KaiABC-based central oscillator proteins can be reconstituted inside a test tube, and the post-translational modification cycle occurs with 24 h periodicity. KaiC's two major phosphorylation sites, Ser-431 and Thr-432, become phosphorylated and dephosphorylated by interacting with KaiA and KaiB, respectively. Here, we mutate Thr-432 into Ser to find the oscillatory phosphoryl transfer reaction damps. Previously, this mutant KaiC was reported to be arrhythmic in vivo. However, we found that the mutant KaiC gradually loses the ability to run in an autonomous manner and stays constitutively phosphorylated after 3 cycles in vitro.

| S-EPMC10061519 | biostudies-literature

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme.

Project description:Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron-electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.

| S-EPMC7683729 | biostudies-literature

Mechanistic insights into the phosphoryl transfer reaction in cyclin-dependent kinase 2: A QM/MM study.

Project description:Cyclin-dependent kinase 2 (CDK2) is an important member of the CDK family exerting its most important function in the regulation of the cell cycle. It catalyzes the transfer of the gamma phosphate group from an ATP (adenosine triphosphate) molecule to a Serine/Threonine residue of a peptide substrate. Due to the importance of this enzyme, and protein kinases in general, a detailed understanding of the reaction mechanism is desired. Thus, in this work the phosphoryl transfer reaction catalyzed by CDK2 was revisited and studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that the base-assisted mechanism is preferred over the substrate-assisted pathway when one Mg2+ is present in the active site, in agreement with a previous theoretical study. The base-assisted mechanism resulted to be dissociative, with a potential energy barrier of 14.3 kcal/mol, very close to the experimental derived value. An interesting feature of the mechanism is the proton transfer from Lys129 to the phosphoryl group at the second transition state, event that could be helping in neutralizing the charge on the phosphoryl group upon the absence of a second Mg2+ ion. Furthermore, important insights into the mechanisms in terms of bond order and charge analysis were provided. These descriptors helped to characterize the synchronicity of bond forming and breaking events, and to characterize charge transfer effects. Local interactions at the active site are key to modulate the charge distribution on the phosphoryl group and therefore alter its reactivity.

| S-EPMC6726203 | biostudies-literature

Phosphoryl Transfer Reaction Snapshots in Crystals: INSIGHTS INTO THE MECHANISM OF PROTEIN KINASE A CATALYTIC SUBUNIT.

Project description:To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca(2+) ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca(2+) cations with Mg(2+) ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. The present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.

| S-EPMC4505467 | biostudies-literature

Arginine coordination in enzymatic phosphoryl transfer: evaluation of the effect of Arg166 mutations in Escherichia coli alkaline phosphatase.

Project description:Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by approximately 3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 A X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state.

| S-EPMC2587100 | biostudies-literature

Conformational dependence of a protein kinase phosphate transfer reaction.

Project description:Atomic motions and energetics for a phosphate transfer reaction catalyzed by the cAMP-dependent protein kinase are calculated by plane-wave density functional theory, starting from structures of proteins crystallized in both the reactant conformation (RC) and the transition-state conformation (TC). In TC, we calculate that the reactants and products are nearly isoenergetic with a 20-kJ/mol barrier, whereas phosphate transfer is unfavorable by 120 kJ/mol in the RC, with an even higher barrier. With the protein in TC, the motions involved in reaction are small, with only P(gamma) and the catalytic proton moving >0.5 A. Examination of the structures reveals that in the RC the active site cleft is not completely closed and there is insufficient space for the phosphorylated serine residue in the product state. Together, these observations imply that the phosphate transfer reaction occurs rapidly and reversibly in a particular conformation of the protein, and that the reaction can be gated by changes of a few tenths of an angstrom in the catalytic site.

| S-EPMC1255735 | 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