Rapid Kinetic Studies Research Articles

Rapid Iodination Kinetic Studies of o-Toluidine in Aqueous Medium: A Pharmacokinetic Insight from QSAR and Molecular Docking of its Iodo Product with Cytochrome P450

The kinetic data of uncatalyzed rapid iodination of o-toluidine using molecular iodine in aqueous medium was investigated by employing hydrodynamic voltammetry (HV) technique. The frequency factor, activation energy and entropy change accompanying the reaction were evaluated. The reaction followed second order kinetics that had a half-life of 90 s and specific reaction rate 888.87 M s–1 at 22.1 ºC. The iodo product was formed via the green route in the aqueous solvent. The physico-chemical characteristics, lipophilicity, water solubility and pharmacokinetic parameters were derived by QSAR analysis. The docking of the iodo product with cytochrome P450 exhibited steric and physiological complementarity of the ligand-protein interface through hydrogen bonding and pi-pi stacking interactions, indicating that the product exhibited metabolic activity was similar to the existing drugs.

Investigating Autoignition Characteristics of Ammonia/Heptamethylnonane Mixtures Over Wide Pressure Ranges: Rapid Compression Machine Measurements and Kinetic Modeling Study

Investigating Autoignition Characteristics of Ammonia/Heptamethylnonane Mixtures Over Wide Pressure Ranges: Rapid Compression Machine Measurements and Kinetic Modeling Study

Rapid anaerobic culture and reaction kinetic study of anammox bacteria on microfluidic chip

Anammox bacteria are being increasingly investigated as part of an emerging nitrogen removal technology. However, due to the difficulty in culturing, current understanding of their behavior is limited. In this study, anaerobic microfluidic chips were used to study anammox bacteria, showing great advantages over reactors. On-chip fluorescence in situ hybridization (FISH) showed the relative abundance of free form anammox bacteria increased by 56.1 % after one week’s culture, an increase that is three times higher than that of bioreactor (17.1 %). For granular form cultures, the nitrogen removal load reached 2.34 ∼ 2.51 kg-N/(m3·d), which was also substantially higher than the bioreactor (∼1.22 kg-N/(m3·d)). Furthermore, studying the kinetics of nitrite inhibition of granular sludge with different particle sizes (100–900 μm) showed that the maximum ammonia load and the nitrite semi-saturation coefficient noticeably decreased for smaller particle sizes. These results illustrate the usefulness of the microfluidic method for in-depth understanding anammox process and its implementation.

Fluorescent labeling of tRNA for rapid kinetic interaction studies with tRNA-binding proteins.

Transfer RNA (tRNA) plays a critical role during translation and interacts with numerous proteins during its biogenesis, functional cycle and degradation. In particular, tRNA is extensively post-transcriptionally modified by various tRNA modifying enzymes which each target a specific nucleotide at different positions within tRNAs to introduce different chemical modifications. Fluorescent assays can be used to study the interaction between a protein and tRNA. Moreover, rapid mixing fluorescence stopped-flow assays provide insights into the kinetics of the tRNA-protein interaction in order to elucidate the tRNA binding mechanism for the given protein. A prerequisite for these studies is a fluorescently labeled molecule, such as fluorescent tRNA, wherein a change in fluorescence occurs upon protein binding. In this chapter, we discuss the utilization of tRNA modifications in order to introduce fluorophores at particular positions within tRNAs. Particularly, we focus on in vitro thiolation of a uridine at position 8 within tRNAs using the tRNA modification enzyme ThiI, followed by labeling of the thiol group with fluorescein. As such, this fluorescently labeled tRNA is primarily unmodified, with the exception of the thiolation modification to which the fluorophore is attached, and can be used as a substrate to study the binding of different tRNA-interacting factors. Herein, we discuss the example of studying the tRNA binding mechanism of the tRNA modifying enzymes TrmB and DusA using internally fluorescein-labeled tRNA.

An NTP-driven mechanism for the nucleotide addition cycle of Escherichia coli RNA polymerase during transcription.

The elementary steps of transcription as catalyzed by E. coli RNA polymerase during one and two rounds of the nucleotide addition cycle (NAC) were resolved in rapid kinetic studies. Modelling of stopped-flow kinetic data of pyrophosphate release in a coupled enzyme assay during one round of the NAC indicates that the rate of pyrophosphate release is significantly less than that for nucleotide incorporation. Upon modelling of the stopped-flow kinetic data for pyrophosphate release during two rounds of the NAC, it was observed that the presence of the next nucleotide for incorporation increases the rate of release of the first pyrophosphate equivalent; incorrect nucleotides for incorporation had no effect on the rate of pyrophosphate release. Although the next nucleotide for incorporation increases the rate of pyrophosphate release, it is still significantly less than the rate of incorporation of the first nucleotide. The results from the stopped-flow kinetic studies were confirmed by using quench-flow followed by thin-layer chromatography (QF-TLC) with only the first nucleotide for incorporation labeled on the gamma phosphate with 32P to monitor pyrophosphate release. Collectively, the results are consistent with an NTP-driven model for the NAC in which the binding of the next cognate nucleotide for incorporation causes a synergistic conformational change in the enzyme that triggers the more rapid release of pyrophosphate, translocation of the enzyme along the DNA template strand and nucleotide incorporation.

Serial Femtosecond Crystallography Reveals the Role of Water in the One- or Two-Electron Redox Chemistry of Compound I in the Catalytic Cycle of the B-Type Dye-Decolorizing Peroxidase DtpB.

Controlling the reactivity of high-valent Fe(IV)-O catalytic intermediates, Compounds I and II, generated in heme enzymes upon reaction with dioxygen or hydrogen peroxide, is important for function. It has been hypothesized that the presence (wet) or absence (dry) of distal heme pocket water molecules can influence whether Compound I undergoes sequential one-electron additions or a concerted two-electron reduction. To test this hypothesis, we investigate the role of water in the heme distal pocket of a dye-decolorizing peroxidase utilizing a combination of serial femtosecond crystallography and rapid kinetic studies. In a dry distal heme site, Compound I reduction proceeds through a mechanism in which Compound II concentration is low. This reaction shows a strong deuterium isotope effect, indicating that reduction is coupled to proton uptake. The resulting protonated Compound II (Fe(IV)-OH) rapidly reduces to the ferric state, giving the appearance of a two-electron transfer process. In a wet site, reduction of Compound I is faster, has no deuterium effect, and yields highly populated Compound II, which is subsequently reduced to the ferric form. This work provides a definitive experimental test of the hypothesis advanced in the literature that relates sequential or concerted electron transfer to Compound I in wet or dry distal heme sites.

Quantitative Assessment of Structure Reactivity Correlation on Rapid Kinetics of Halogenated Aromatic Compounds: A Hydrodynamic Voltammetry Study

Halogenations are mostly rapid and cannot be studied by the conventional method, hence, hydrodynamic voltammetry which is based on voltammetric principles can be adopted to study these fast reactions, provided either a reactant or a product in the reaction is electro reducible. These reactions are mostly electrophillic substitution reactions and second order when molecular halogens are used as a reagent. Rapid kinetic studies on some important halogenated aromatic compounds at rotating platinum electrode (RPE) using hydrodynamic voltammetry are summarized and reviewed for a period of the last few years. The advancement made to the technique as well as its application for mechanistic purposes, electrode kinetic measurements, pharmaceutical, and analytical applications have been addressed. As an investigative tool, kinetics has been used to obtain thermodynamic data that allows a quantitative comparison of the reactivity of the two substrates such as activation energy, velocity constants, frequency factors, and entropy change. This aids in configuring the mechanistic route, from among those theoretically possible by checking the compatibility with experimentally observed facts.

An atomistic simulation study of rapid solidification kinetics and crystal defects in dilute Al–Cu alloys

Rapid solidification kinetics of dilute Al–Cu alloys is simulated using a quantum mechanics based bond-order potential (BOP), in free solidification conditions, to determine kinetic and thermodynamic properties of solidification, as well as point defects and chemical ordering of the solidified structures. We measure the anisotropic kinetic coefficient, anisotropic solid–liquid interface energy, as well as solute trapping kinetics in terms of partition coefficient versus velocity and solute drag coefficient. Furthermore, solid–liquid interface free energy and its anisotropy are measured in equilibrium simulations, showing reasonably good agreement with previous studies. We also verified the self-consistency of the MD simulations, by comparing the interfacial temperature vs. velocity to that predicted by the continuous growth model. These solid–liquid interface properties are important for quantitative parametrization of larger scale solidification modeling techniques such as phase field models. We also investigated the point defect content, local chemical ordering, and local crystalline structures in the rapidly solidified samples. We found clustering of solute with vacancies, whereas copper atoms repelled each other in these dilute alloy simulations. In addition to vacancies, a large number of interstitials were found. In solidification velocities approaching the complete solute trapping regime, we found that the vacancies and interstitials formed in conjunction, i.e. as Frenkel pairs. Finally, in addition to FCC, we detected BCC and HCP phases, where the latter two were accompanied by an increase in local copper content. Understanding the formation of point defects and their relationship to chemical ordering is an important step towards controlling the formation of pre-precipitates and precipitates, which are an important strengthening mechanism for aluminum–copper alloys.

Synthesis of hydrous CeO2 polypyrrole nanocomposite as a rapid and efficient adsorbent for defluoridation of drinking water

Contamination of water by fluoride ions is one of the major threats to human life and thus must be removed from drinking water. A novel CeO2 polypyrrole nanocomposite (HCeO2@PPy) was fabricated by co-precipitation of CeO2 nanoparticles followed by in situ chemical oxidative polymerization. The as-synthesised adsorbent was characterized using a variety of physico-chemical techniques which included Fourier Transform Infrared Spectroscopy (FTIR), X-Ray diffraction (XRD), Braunauer-Emmett-Teller (BET), Field Emission Scanning Electron Microscopy/Energy Dispersive (FE-SEM/EDS), High Resolution-Transmission Electron Microscopy (HR-TEM), X-ray Photoelectron Spectroscopy (XPS) and point-of-zero charge (pzc) determination. The HCeO2@PPy nanocomposite adsorbent was applied for the removal of F− ions from aqueous solution. Application of the adsorption modelling, revealed that the Langmuir adsorption isotherm model best described the process with a maximum adsorption capacity of 70.92 mg g−1, at an optimum pH of 6.0 ± 0.2, 0.03 g adsorbent dose and 25 °C. Rapid kinetic studies revealed that the pseudo-second-order model gives a better description of the adsorption process. Thermodynamic data showed that the adsorption process was physical, spontaneous and feasible. The behaviour of the amino functional groups within HCeO2@PPy moiety and the nanometal oxide surface hydroxides at different pHs along with the pHpzc, FTIR and XPS spectra analyses were used to explain the mechanism of adsorption. The mechanism was conceived to be anionic exchange of hydroxyl group with the F− ions and electrostatic attractions of protonated hydroxyl on the surface of the adsorbent as well as the nitrogen atoms of amino groups from the polypyrrole (PPy) moiety with F− ions. The determined thermodynamic parameters, enthalpy change (ΔH° = −26.33 kJmol−1) and Gibbs free energy change (ΔG° = −19.074 to −10.028 kJ mol−1) indicated the exothermic and spontaneous nature of the sorption process. Further evaluation on the as-prepared adsorbent exposed a moderately selective material which exhibited excellent removal ability of F− ions from ground water at its natural pH to below WHO stipulated levels with an adsorption-desorption efficiency of up to three cycles. Therefore, hydrous CeO2 polypyrrole nanocomposite has revealed great potential for water defluoridation.

A Novel High-Throughput FLIPR Tetra-Based Method for Capturing Highly Confluent Kinetic Data for Structure-Kinetic Relationship Guided Early Drug Discovery.

Target engagement by small molecules is necessary for producing a physiological outcome. In the past, a lot of emphasis was placed on understanding the thermodynamics of such interactions to guide structure-activity relationships. It is becoming clearer, however, that understanding the kinetics of the interaction between a small-molecule inhibitor and the biological target [structure-kinetic relationship (SKR)] is critical for selection of the optimum candidate drug molecule for clinical trial. However, the acquisition of kinetic data in a high-throughput manner using traditional methods can be labor intensive, limiting the number of molecules that can be tested. As a result, in-depth kinetic studies are often carried out on only a small number of compounds, and usually at a later stage in the drug discovery process. Fundamentally, kinetic data should be used to drive key decisions much earlier in the drug discovery process, but the throughput limitations of traditional methods preclude this. A major limitation that hampers acquisition of high-throughput kinetic data is the technical challenge in collecting substantially confluent data points for accurate parameter estimation from time course analysis. Here, we describe the use of the fluorescent imaging plate reader (FLIPR), a charge-coupled device (CCD) camera technology, as a potential high-throughput tool for generating biochemical kinetic data with smaller time intervals. Subsequent to the design and optimization of the assay, we demonstrate the collection of highly confluent time-course data for various kinase protein targets with reasonable throughput to enable SKR-guided medicinal chemistry. We select kinase target 1 as a special case study with covalent inhibition, and demonstrate methods for rapid and detailed analysis of the resultant kinetic data for parameter estimation. In conclusion, this approach has the potential to enable rapid kinetic studies to be carried out on hundreds of compounds per week and drive project decisions with kinetic data at an early stage in drug discovery.

Crystallographic and kinetic analyses of the FdsBG subcomplex of the cytosolic formate dehydrogenase FdsABG from Cupriavidus necator

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH-, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

Synthesis of a robust layered metal sulfide for rapid and effective removal of Sr2+ from aqueous solutions

A novel robust metal sulfide (NaTS) was synthesized via a simple one-step hydrothermal reaction for the first time. SEM, TEM, EDS, and XPS analyses indicated that NaTS has a sheet-like structure stacked by hexagonal thin flakes with the formula Na2Sn3S7. PXRD and TG results indicated that NaTS was chemically and thermally stable. The rapid kinetic studies showed that NaTS can rapidly remove Sr2+, with a rate of 98.71% in the first minute, and can reach adsorption equilibrium within 5 min. A pseudo-second-order model and intraparticle diffusion model were found to be good fits for the NaTS adsorption data. The adsorption isotherms were well described by the Langmuir model and NaTS exhibited high adsorption capacity for Sr2+ (80.0 mg/g at 298 K). They also displayed a high distribution coefficient (Kd = 3.43 × 107 mL/g) and excellent removal efficiency of 99.98% with 0.5 g/L dosage in 5 mg/L Sr2+ solutions. In particular, NaTS exhibited outstanding affinity (Kd ≥ 1.31 × 105 mL/g) for Sr2+ over a broad pH range of 3–13. In addition, the adsorption mechanism of NaTS towards Sr2+ was ion exchange and Sr⋯S binding, as confirmed by EDS, XPS and ICP-OES. Furthermore, NaTS had significant advantages for Sr2+ removal compared with other adsorbents. In general, these adsorption advantages combined with facile synthesis, as well as structural stability, make NaTS promising for removal of radioactive Sr2+ from strongly acidic or alkaline radioactive wastewaters easily and efficiently.

Kinetic Profiling of the Hydrolytic Reaction of Benazepril: Metabolic Pathway Simulation.

A simple, specific, and rapid kinetic study of benazepril (BNZ) hydrolysis was developed and validated using HPLC. BNZ was degraded using 0.1 N sodium hydroxide at room temperature to produce benazeprilat, which is an active metabolite of BNZ and acts as an angiotensin-converting enzyme inhibitor. Analysis was carried out using an Athena C18 column (4.6 × 250 mm, 5 µm particle size). The mobile phase consists of a mixture of phosphate buffer (pH 4.5) and acetonitrile (53 + 47, v/v) at a flow rate of 1 mL/min. UV detection was accomplished at 242 nm using moexipril as the internal standard. The method was validated according to International Conference on Harmonization guidelines, and the calibration curve was linear over the range 10-100 µg/mL, with acceptable accuracy and precision. Kinetic profiling of the hydrolysis was shown to follow pseudo-first-order kinetics. The method was applied to the assay of BNZ in combined dosage form with no interference from other ingredients. The obtained results were statistically compared with those of the official method, showing no significant difference.

Cooperative Interactions of Three Hotspot Heparin Binding Residues Are Critical for Allosteric Activation of Antithrombin by Heparin.

Heparin allosterically activates the anticoagulant serpin, antithrombin, by binding through a sequence-specific pentasaccharide and inducing activating conformational changes in the protein. Three basic residues of antithrombin, Lys114, Lys125, and Arg129, have been shown to be hotspots for binding the pentasaccharide, but the molecular basis for such hotspot binding has been unclear. To determine whether this results from cooperative interactions, we analyzed the effects of single, double, and triple mutations of the hotspot residues on pentasaccharide binding and activation of antithrombin. Double-mutant cycles revealed that the contribution of each residue to pentasaccharide binding energy was progressively reduced when one or both of the other residues were mutated, indicating strong coupling between each pair of residues that was dependent on the third residue and reflective of the three residues acting as a cooperative unit. Rapid kinetic studies showed that the hotspot residue mutations progressively abrogated the ability of the pentasaccharide to bind productively to native antithrombin and to conformationally activate the serpin by engaging the hotspot residues in an induced-fit interaction. Examination of the antithrombin-pentasaccharide complex structure revealed that the hotspot residues form two adjoining binding pockets for critical sulfates of the pentasaccharide that structurally link these residues. Together, these findings demonstrate that cooperative interactions of Lys114, Lys125, and Arg129 are critical for the productive induced-fit binding of the heparin pentasaccharide to antithrombin that allosterically activates the anticoagulant function of the serpin.

Structural, Kinetic, and Computational Characterization of the Elusive Arylpalladium(II)boronate Complexes in the Suzuki-Miyaura Reaction.

The existence of the oft-invoked intermediates containing the crucial Pd-O-B subunit, the "missing link", has been established in the Suzuki-Miyaura cross-coupling reaction. The use of low-temperature, rapid injection NMR spectroscopy (RI-NMR), kinetic studies, and computational analysis has enabled the generation, observation, and characterization of these highly elusive species. The ability to confirm the intermediacy of Pd-O-B-containing species provided the opportunity to clarify mechanistic aspects of the transfer of the organic moiety from boron to palladium in the key transmetalation step. Specifically, these studies establish the identity of two different intermediates containing Pd-O-B linkages, a tri-coordinate (6-B-3) boronic acid complex and a tetra-coordinate (8-B-4) boronate complex, both of which undergo transmetalation leading to the cross-coupling product. Two distinct mechanistic pathways have been elucidated for stoichiometric reactions of these complexes: (1) transmetalation via an unactivated 6-B-3 intermediate that dominates in the presence of an excess of ligand, and (2) transmetalation via an activated 8-B-4 intermediate that takes place with a deficiency of ligand.

Mechanism of Lagging-Strand DNA Replication in Eukaryotes.

This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA polymerase α-primase (Pol α) provides insights how each of the millions of Okazaki fragments in a mammalian cell is primed by the primase subunit and further extended by its polymerase subunit. Rapid kinetic studies of Okazaki fragment elongation by Pol δ illuminate events when the polymerase encounters the double-stranded RNA-DNA block of the preceding Okazaki fragment. This block acts as a progressive molecular break that provides both time and opportunity for the flap endonuclease 1 (FEN1) to access the nascent flap and cut it. The iterative action of Pol δ and FEN1 is coordinated by the replication clamp PCNA and produces a regulated degradation of the RNA primer, thereby preventing the formation of long-strand displacement flaps. Occasional long flaps are further processed by backup nucleases including Dna2.

Characterization of a new Baeyer-Villiger monooxygenase and conversion to a solely N-or S-oxidizing enzyme by a single R292 mutation

BackgroundAr-BVMO is a recently discovered Baeyer-Villiger monooxygenase from the genome of Acinetobacter radioresistens S13 closely related to medically relevant ethionamide monooxygenase EtaA (prodrug activator) and capable of inactivating the imipenem antibiotic. MethodsThe co-substrate preference as well as steady-state and rapid kinetics studies of the recombinant purified protein were carried out using stopped-flow spectroscopy under anaerobic and aerobic conditions. Kd values were measured by isothermal calorimetry. Enzymatic activity was determined by measuring the amount of product formed using high pressure liquid chromatography or gas chromatography. Site-directed mutagenesis experiments were performed to decipher the role of the active site arginine-292. ResultsAr-BVMO was found to oxidize ethionamide as well as linear ketones. Mechanistic studies on the wild type enzyme using stopped-flow spectroscopy allowed for the detection of the characteristic oxygenating C4a-(hydro)peroxyflavin intermediate, which decayed rapidly in the presence of the substrate. Replacement of arginine 292 in Ar-BVMO by glycine or alanine resulted in greatly reduced or no Baeyer-Villiger activity, respectively, demonstrating the crucial role of this residue in catalysis of ketone substrates. However, both the R292A and R292G mutants are capable of carrying out N- and S-oxidation reactions. ConclusionsSubstrate profiling of Ar-BVMO confirms its close relationship to EtaA; ethionamide is one of its substrates. The active site Arginine 292 is required for its Baeyer-Villiger activity but not for heteroatom oxidation. General significanceA single mutation converts Ar-BVMO to a unique S- or N-monooxygenase, a useful biocatalyst for the production of oxidized metabolites of human drug metabolizing enzymes.

Pre-transmetalation intermediates in the Suzuki-Miyaura reaction revealed: The missing link.

Despite the widespread application of Suzuki-Miyaura cross-coupling to forge carbon-carbon bonds, the structure of the reactive intermediates underlying the key transmetalation step from the boron reagent to the palladium catalyst remains uncertain. Here we report the use of low-temperature rapid injection nuclear magnetic resonance spectroscopy and kinetic studies to generate, observe, and characterize these previously elusive complexes. Specifically, this work establishes the identity of three different species containing palladium-oxygen-boron linkages, a tricoordinate boronic acid complex, and two tetracoordinate boronate complexes with 2:1 and 1:1 stoichiometry with respect to palladium. All of these species transfer their boron-bearing aryl groups to a coordinatively unsaturated palladium center in the critical transmetalation event.

Structural and kinetic studies on RosA, the enzyme catalysing the methylation of 8-demethyl-8-amino-d-riboflavin to the antibiotic roseoflavin.

N,N‐8‐demethyl‐8‐amino‐d‐riboflavin dimethyltransferase (RosA) catalyses the final dimethylation of 8‐demethyl‐8‐amino‐d‐riboflavin (AF) to the antibiotic roseoflavin (RoF) in Streptomyces davawensis. In the present study, we solved the X‐ray structure of RosA, and determined the binding properties of substrates and products. Moreover, we used steady‐state and rapid reaction kinetic studies to obtain detailed information on the reaction mechanism. The structure of RosA was found to be similar to that of previously described S‐adenosylmethionine (SAM)‐dependent methyltransferases, featuring two domains: a mainly α‐helical ‘orthogonal bundle’ and a Rossmann‐like domain (α/β twisted open sheet). Bioinformatics studies and molecular modelling enabled us to predict the potential SAM and AF binding sites in RosA, suggesting that both substrates, AF and SAM, bind independently to their respective binding pocket. This finding was confirmed by kinetic experiments that demonstrated a random‐order ‘bi‐bi’ reaction mechanism. Furthermore, we determined the dissociation constants for substrates and products by either isothermal titration calorimetry or UV/Vis absorption spectroscopy, revealing that both products, RoF and S‐adenosylhomocysteine (SAH), bind more tightly to RosA compared with the substrates, AF and SAM. This suggests that RosA may contribute to roseoflavin resistance in S. davawensis. The tighter binding of products is also reflected by the results of inhibition experiments, in which RoF and SAH behave as competitive inhibitors for AF and SAM, respectively. We also showed that formation of a ternary complex of RosA, RoF and SAH (or SAM) leads to drastic spectral changes that are indicative of a hydrophobic environment.DatabaseStructural data are available in the Protein Data Bank under accession number 4D7K.

The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome.

Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.