Browse
Submit Data
Databases
API
Help

Dataset Information

18 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Examinations of the Chemical Step in Enzyme Catalysis.

ABSTRACT: Advances in computational and experimental methods in enzymology have aided comprehension of enzyme-catalyzed chemical reactions. The main difficulty in comparing computational findings to rate measurements is that the first examines a single energy barrier, while the second frequently reflects a combination of many microscopic barriers. We present here intrinsic kinetic isotope effects and their temperature dependence as a useful experimental probe of a single chemical step in a complex kinetic cascade. Computational predictions are tested by this method for two model enzymes: dihydrofolate reductase and thymidylate synthase. The description highlights the significance of collaboration between experimentalists and theoreticians to develop a better understanding of enzyme-catalyzed chemical conversions.

SUBMITTER: Singh P

PROVIDER: S-EPMC4978192 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Examinations of the Chemical Step in Enzyme Catalysis.

Singh P P Islam Z Z Kohen A A

Methods in enzymology 20160628

Advances in computational and experimental methods in enzymology have aided comprehension of enzyme-catalyzed chemical reactions. The main difficulty in comparing computational findings to rate measurements is that the first examines a single energy barrier, while the second frequently reflects a combination of many microscopic barriers. We present here intrinsic kinetic isotope effects and their temperature dependence as a useful experimental probe of a single chemical step in a complex kinetic ...[more]

PMID: 27498642

Similar Datasets

A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis.

Project description:Conformational dynamics play a key role in enzyme catalysis. Although protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of Escherichia coli dihydrofolate reductase that abrogates millisecond-time-scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond time scale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for design of novel protein catalysts.

| S-EPMC3151171 | biostudies-literature

Machine Learning Identifies Chemical Characteristics That Promote Enzyme Catalysis.

Project description:Despite tremendous progress in understanding and engineering enzymes, knowledge of how enzyme structures and their dynamics induce observed catalytic properties is incomplete, and capabilities to engineer enzymes fall far short of industrial needs. Here, we investigate the structural and dynamic drivers of enzyme catalysis for the rate-limiting step of the industrially important enzyme ketol-acid reductoisomerase (KARI) and identify a region of the conformational space of the bound enzyme-substrate complex that, when populated, leads to large increases in reactivity. We apply computational statistical mechanical methods that implement transition interface sampling to simulate the kinetics of the reaction and combine this with machine learning techniques from artificial intelligence to select features relevant to reactivity and to build predictive models for reactive trajectories. We find that conformational descriptors alone, without the need for dynamic ones, are sufficient to predict reactivity with greater than 85% accuracy (90% AUC). Key descriptors distinguishing reactive from almost-reactive trajectories quantify substrate conformation, substrate bond polarization, and metal coordination geometry and suggest their role in promoting substrate reactivity. Moreover, trajectories constrained to visit a region of the reactant well, separated from the rest by a simple hyperplane defined by ten conformational parameters, show increases in computed reactivity by many orders of magnitude. This study provides evidence for the existence of reactivity promoting regions within the conformational space of the enzyme-substrate complex and develops methodology for identifying and validating these particularly reactive regions of phase space. We suggest that identification of reactivity promoting regions and re-engineering enzymes to preferentially populate them may lead to significant rate enhancements.

| S-EPMC6407039 | biostudies-literature

Chemical complementation: a reaction-independent genetic assay for enzyme catalysis.

Project description:A high-throughput assay for enzyme activity has been developed that is reaction independent. In this assay, a small-molecule yeast three-hybrid system is used to link enzyme catalysis to transcription of a reporter gene in vivo. Here we demonstrate the feasibility of this approach by using a well-studied enzyme-catalyzed reaction, cephalosporin hydrolysis by the Enterobacter cloacae P99 cephalosporinase (beta-lactam hydrolase, EC ). We show that the three-hybrid system can be used to read out cephalosporinase activity in vivo as a change in the level of transcription of a lacZ reporter gene and that the wild-type cephalosporinase can be isolated from a pool of inactive mutants by using a lacZ screen. The assay has been designed so that it can be applied to different chemical reactions without changing the components of the three-hybrid system. A reaction-independent high-throughput assay for protein function should be a powerful tool for protein engineering and enzymology, drug discovery, and proteomics.

| S-EPMC139179 | biostudies-literature

Enzyme millisecond conformational dynamics do not catalyze the chemical step.

Project description:The idea that enzymes catalyze reactions by dynamical coupling between the conformational motions and the chemical coordinates has recently attracted major experimental and theoretical interest. However, experimental studies have not directly established that the conformational motions transfer energy to the chemical coordinate, and simulating enzyme catalysis on the relevant timescales has been impractical. Here, we introduce a renormalization approach that transforms the energetics and dynamics of the enzyme to an equivalent low-dimensional system, and allows us to simulate the dynamical coupling on a ms timescale. The simulations establish, by means of several independent approaches, that the conformational dynamics is not remembered during the chemical step and does not contribute significantly to catalysis. Nevertheless, the precise nature of this coupling is a question of great importance.

| S-EPMC2762662 | biostudies-literature

On the relationship between folding and chemical landscapes in enzyme catalysis.

Project description:Elucidating the relationship between the folding landscape of enzymes and their catalytic power has been one of the challenges of modern enzymology. The present work explores this issue by using a simplified folding model to generate the free-energy landscape of an enzyme and then to evaluate the activation barriers for the chemical step in different regions of the landscape. This approach is used to investigate the recent finding that an engineered monomeric chorismate mutase exhibits catalytic efficiency similar to the naturally occurring dimer even though it exhibits the properties of an intrinsically disordered molten globule. It is found that the monomer becomes more confined than its native-like counterpart upon ligand binding but still retains a wider catalytic region. Although the overall rate acceleration is still determined by reduction of the reorganization energy, the detailed contribution of different barriers yields a more complex picture for the chemical process than that of a single path. This work provides insight into the relationship between folding landscapes and catalysis. The computational approach used here may also provide a powerful strategy for modeling single-molecule experiments and designing enzymes.

| S-EPMC2544547 | biostudies-literature

Prolyl endopeptidase catalysis. A physical rather than a chemical step is rate-limiting.

Project description:Prolyl endopeptidase represents a new family of serine proteases, and it has a mechanistic feature distinct from that of the enzymes of the extensively studied chymotrypsin and subtilisin families. The rate-determining step in the catalysis of serine proteases is a general base/acid-catalysed chemical step. For prolyl endopeptidase, however, the chemical step is not rate-limiting, as demonstrated by using substrates with different leaving groups. It is known that the acylation of chymotrypsin and subtilisin proceeds faster by several orders of magnitude with the activated nitrophenyl ester than with the corresponding amide substrates. In contrast, for the acylation of prolyl endopeptidase similar rate constants were obtained with nitrophenyl ester and several amide substrates. This result, combined with kinetic isotope studies [Polgár (1991) Eur. J. Biochem. 197, 441-447], offers strong evidence that a physical step, presumably a conformational change associated with substrate binding, is the rate-determining step in the prolyl endopeptidase catalysis.

| S-EPMC1130933 | biostudies-other

Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1.

Project description:Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and 20α,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. Herein, we present the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the "soft" recognition of the 22HC 3β-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1-22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s.

| S-EPMC3037674 | biostudies-literature

Kinetic Detection of Orthogonal Protein and Chemical Coordinates in Enzyme Catalysis: Double Mutants of Soybean Lipoxygenase.

Project description:Soybean lipoxygenase-1 (SLO-1) is a paradigmatic enzyme system for studying the contribution of hydrogen tunneling to enzymatic proton-coupled electron transfer processes. In this study, the impact of pairs of double mutants on the properties of SLO-1 is presented. Steady-state rates and their deuterium kinetic isotope effects (KIEs) have been measured for the bimolecular reaction of enzyme with free substrate (kcat/Km) and compared to the unimolecular rate constant, kcat. A key kinetic finding is that the competitive KIEs on the second-order rate constant (kcat/Km) are all reduced from (D)kcat and, despite large changes in rate and activation parameters, remain essentially unaltered under a variety of conditions. These data implicate a protein reaction coordinate that is orthogonal to the chemical reaction coordinate and controls the concentration of the active enzyme. This study introduces a new means to interrogate the alteration of conformational landscapes that can occur following site-specific mutagenesis.

| S-EPMC4565126 | biostudies-literature

Substrate catalysis enhances single-enzyme diffusion.

Project description:We show that diffusion of single urease enzyme molecules increases in the presence of urea in a concentration-dependent manner and calculate the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concentrations ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local molecular pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme molecule and lay the foundation for a further understanding of biological force generation and the development of enzyme-driven nanomotors.

| S-EPMC2832858 | biostudies-literature

Linking protein motion to enzyme catalysis.

Project description:Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate binding, catalysis of the chemical conversion, and product release. The relationship between protein motions and catalytic activity is of contemporary interest in enzymology. To understand the factors influencing the rates of enzyme-catalyzed reactions, the dynamics of the protein-solvent-ligand complex must be considered. The current review presents two case studies of enzymes-dihydrofolate reductase (DHFR) and thymidylate synthase (TSase)-and discusses the role of protein motions in their catalyzed reactions. Specifically, we will discuss the utility of kinetic isotope effects (KIEs) and their temperature dependence as tools in probing such phenomena.

| S-EPMC4341894 | 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