Showing papers on "Substrate (chemistry) published in 2013"

Modifying enzyme activity and selectivity by immobilization.

Abstract: Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.

A Powerful Aluminum Catalyst for the Synthesis of Highly Functional Organic Carbonates

Abstract: An aluminum complex based on an amino triphenolate ligand scaffold shows unprecedented high activity (initial TOFs up to 36 000 h–1), broad substrate scope, and functional group tolerance in the formation of highly functional organic carbonates prepared from epoxides and CO2. The developed catalytic protocol is further characterized by low catalyst loadings and relative mild reaction conditions using a cheap, abundant, and nontoxic metal.

High-throughput fluorometric measurement of potential soil extracellular enzyme activities.

Abstract: Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample). Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.

Catalyst-Controlled Aliphatic C–H Oxidations with a Predictive Model for Site-Selectivity

Abstract: Selective aliphatic C-H bond oxidations may have a profound impact on synthesis because these bonds exist across all classes of organic molecules. Central to this goal are catalysts with broad substrate scope (small-molecule-like) that predictably enhance or overturn the substrate’s inherent reactivity preference for oxidation (enzyme-like). We report a simple small-molecule, non-heme iron catalyst that achieves predictable catalyst-controlled site-selectivity in preparative yields over a range of topologically diverse substrates. A catalyst reactivity model quantitatively correlates the innate physical properties of the substrate to the site-selectivities observed as a function of the catalyst.

Comprehensive Enzyme Kinetics

Abstract: 1. Introduction. 2. Chemical kinetics. 3. Kinetics of monosubstrate reactions. 4. Derivations of rate equations. 5. Linear inhibition. 6. Hyperbolic and parabolic inhibition. 7. Enzyme activation. 8. Kinetics of rapid equilibrium bisubstrate reactions. 9. Steady state kinetics of bisubstrate reactions. 10. Kinetic analysis of bisubstrate mechanisms. 11. Substrate inhibition and mixed dead-end and product inhibition. 12. Kinetics of trisubstrate reactions. 13. Cooperative and allosteric effects. 14. The pH dependence of enzyme catalysis. 15. Effects of temperature on enzyme reactions. 16. Isotope exchange. 17. Solvent and kinetic isotope effects. 18. Statistical analysis of initial rate and binding data. Subject index.

Psychrophilic enzymes: from folding to function and biotechnology.

Abstract: Psychrophiles thriving permanently at near-zero temperatures synthesize cold-active enzymes to sustain their cell cycle. Genome sequences, proteomic, and transcriptomic studies suggest various adaptive features to maintain adequate translation and proper protein folding under cold conditions. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Several open questions in the field are also highlighted.

Enzyme molecules as nanomotors.

Abstract: Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme molecules increase in the presence of the substrate, hydrogen peroxide, in a concentration-dependent manner. Employing a microfluidic device to generate a substrate concentration gradient, we show that both catalase and urease enzyme molecules spread toward areas of higher substrate concentration, a form of chemotaxis at the molecular scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chemically interconnected enzymes can be drawn together.

Iron(IV)hydroxide pKa and the Role of Thiolate Ligation in C–H Bond Activation by Cytochrome P450

Abstract: Cytochrome P450 enzymes activate oxygen at heme iron centers to oxidize relatively inert substrate carbon-hydrogen bonds. Cysteine thiolate coordination to iron is posited to increase the pK(a) (where K(a) is the acid dissociation constant) of compound II, an iron(IV)hydroxide complex, correspondingly lowering the one-electron reduction potential of compound I, the active catalytic intermediate, and decreasing the driving force for deleterious auto-oxidation of tyrosine and tryptophan residues in the enzyme's framework. Here, we report on the preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥90% yield. Using rapid mixing technologies in conjunction with Mossbauer, ultraviolet/visible, and x-ray absorption spectroscopies, we determine a pK(a) value for this compound of 11.9. Marcus theory analysis indicates that this elevated pK(a) results in a >10,000-fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

A nanosized metal–organic framework of Fe-MIL-88NH2 as a novel peroxidase mimic used for colorimetric detection of glucose

Abstract: In this paper, a nanosized porous metal–organic framework, Fe-MIL-88NH2, was facilely prepared with a uniform octahedral shape by the addition of acetic acid, and for the first time was demonstrated to possess intrinsic peroxidase-like activity. Kinetic analysis and electron spin resonance measurements indicated that the catalytic behavior was consistent with typical Michaelis–Menten kinetics and follows a ping-pong mechanism. As a novel peroxidase mimic material, Fe-MIL-88NH2 shows the advantages of high catalytic efficiency, ultrahigh stability and high biocompatibility in aqueous medium compared with natural enzymes and other peroxidase nanomimetics. Here, Fe-MIL-88NH2 was used to quickly catalyze oxidation of the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a colored product, which provided a simple, sensitive and selective method for the colorimetric detection of glucose. Glucose could be linearly detected in the range from 2.0 × 10−6 to 3.0 × 10−4 M with a detection limit of 4.8 × 10−7 M, and the color variation for glucose response was also obvious by visual observation at concentrations as low as 2.0 × 10−6 M. More importantly, the colorimetric method could be successfully applied to the determination of glucose in diluted serum samples.

Tuning the Electronic and Chemical Properties of Monolayer MoS2 Adsorbed on Transition Metal Substrates

Abstract: Using first-principles calculations within density functional theory, we investigate the electronic and chemical properties of a single-layer MoS2 adsorbed on Ir(111), Pd(111), or Ru(0001), three representative transition metal substrates having varying work functions but each with minimal lattice mismatch with the MoS2 overlayer. We find that, for each of the metal substrates, the contact nature is of Schottky-barrier type, and the dependence of the barrier height on the work function exhibits a partial Fermi-level pinning picture. Using hydrogen adsorption as a testing example, we further demonstrate that the introduction of a metal substrate can substantially alter the chemical reactivity of the adsorbed MoS2 layer. The enhanced binding of hydrogen, by as much as ∼0.4 eV, is attributed in part to a stronger H–S coupling enabled by the transferred charge from the substrate to the MoS2 overlayer, and in part to a stronger MoS2-metal interface by the hydrogen adsorption. These findings may prove to be ins...

Graphene‐Veiled Gold Substrate for Surface‐Enhanced Raman Spectroscopy

Abstract: Surface-enhanced Raman spectroscopy (SERS) [ 1–3 ] can boost the pristine Raman signal by 10 8 times or more, which has exhibited amazing potential for ultrasensitive analytical applications. However, the inherent complexity of a SERS system makes this a “double-edged sword”. The high sensitivity of SERS (even capable for detection of a single molecule [ 4 , 5 ] ) is dimmed by its poor reproducibility (which makes it diffi cult for peak-to-peak assignment of each vibrational mode). By the virtue of wide achievements on miscellaneous substrate preparation methods, both the understanding of the enhancement mechanism of SERS and the pursuit of more desirable SERS signals have taken impressive steps forward. Nevertheless, there remains more to be further investigated. [ 6 , 7 ] To improve signal cleanliness against substrate-induced fl uctuations and to investigate the detailed SERS activity–morphology relationship are two important aspects, both of which are critical for “out of the laboratory” applications of SERS. In the present study, we exploited graphene to fabricate a veiled, rough, gold substrate for SERS. The graphene-veiled gold substrate inherits the concept of metal–molecule isolation, [ 8 , 9 ] providing a passivated surface for SERS which exhibits good signal reproducibility. By tuning the morphology of the gold–graphene combined structure, the detailed electromagnetic enhancement activity–morphology relationship is investigated. Actually, among the various concerns about the performance of a SERS substrate, the issue of metal–molecule contact induced signal variations has become a subject with rising importance. [ 8 , 9 ] The main unfavourable disturbances include chemical adsorption-induced vibrations, charge transfer between the metal and molecules, photo-induced damage and metal-catalyzed side reactions, etc. [ 6 ] Using a thin and pinholefree layer of SiO 2 or Al 2 O 3 as an inert shell, [ 8 ] Tian’s group demonstrated a series of measurements which are challenging with a normal metal substrate. Fabrication of SERS substrates with a passivated surface at a lowest loss of electromagnetic

Laccase versus laccase-like multi-copper oxidase: a comparative study of similar enzymes with diverse substrate spectra.

Abstract: Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Owing to their broad substrate range, copper-containing laccases are versatile biocatalysts, capable of oxidizing numerous natural and non-natural industry-relevant compounds, with water as the sole by-product. In the present study, 10 of the 11 multi-copper oxidases, hitherto considered to be laccases, from fungi, plant and bacterial origin were compared. A substrate screen of 91 natural and non-natural compounds was recorded and revealed a fairly broad but distinctive substrate spectrum amongst the enzymes. Even though the enzymes share conserved active site residues we found that the substrate ranges of the individual enzymes varied considerably. The EC classification is based on the type of chemical reaction performed and the actual name of the enzyme often refers to the physiological substrate. However, for the enzymes studied in this work such classification is not feasible, even more so as their prime substrates or natural functions are mainly unknown. The classification of multi-copper oxidases assigned as laccases remains a challenge. For the sake of simplicity we propose to introduce the term “laccase-like multi-copper oxidase” (LMCO) in addition to the term laccase that we use exclusively for the enzyme originally identified from the sap of the lacquer tree Rhus vernicifera.

Chemiluminescent Detection of Enzymatically Produced Hydrogen Sulfide: Substrate Hydrogen Bonding Influences Selectivity for H2S over Biological Thiols

Abstract: Hydrogen sulfide (H2S) is now recognized as an important biological regulator and signaling agent that is active in many physiological processes and diseases. Understanding the important roles of this emerging signaling molecule has remained challenging, in part due to the limited methods available for detecting endogenous H2S. Here we report two reaction-based ChemiLuminescent Sulfide Sensors, CLSS-1 and CLSS-2, with strong luminescence responses toward H2S (128- and 48-fold, respectively) and H2S detection limits (0.7 ± 0.3, 4.6 ± 2.0 μM, respectively) compatible with biological H2S levels. CLSS-2 is highly selective for H2S over other reactive sulfur, nitrogen, and oxygen species (RSONS) including GSH, Cys, Hcy, S2O32–, NO2–, HNO, ONOO–, and NO. Despite its similar chemical structure, CLSS-1 displays lower selectivity toward amino acid-derived thiols than CLSS-2. The origin of this differential selectivity was investigated using both computational DFT studies and NMR experiments. Our results suggest a ...

Mild Copper-Mediated Fluorination of Aryl Stannanes and Aryl Trifluoroborates

Abstract: This communication describes a mild copper-mediated fluorination of aryl stannanes and aryl trifluoroborates with N-fluoro-2,4,6-trimethylpyridinium triflate. This protocol demonstrates broad substrate scope and functional group tolerance, and does not require the use of any noble metal additives. The reaction is proposed to proceed via an arylcopper(III) fluoride intermediate.

Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis

Abstract: Substrate binding is typically one of the rate-limiting steps preceding enzyme catalytic action during homogeneous reactions. However, interfacial-based enzyme catalysis on insoluble crystalline substrates, like cellulose, has additional bottlenecks of individual biopolymer chain decrystallization from the substrate interface followed by its processive depolymerization to soluble sugars. This additional decrystallization step has ramifications on the role of enzyme–substrate binding and its relationship to overall catalytic efficiency. We found that altering the crystalline structure of cellulose from its native allomorph Iβ to IIII results in 40–50% lower binding partition coefficient for fungal cellulases, but surprisingly, it enhanced hydrolytic activity on the latter allomorph. We developed a comprehensive kinetic model for processive cellulases acting on insoluble substrates to explain this anomalous finding. Our model predicts that a reduction in the effective binding affinity to the substrate coupled with an increase in the decrystallization procession rate of individual cellulose chains from the substrate surface into the enzyme active site can reproduce our anomalous experimental findings.

Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens P-001

Abstract: Amylases are one of the most important enzymes in present-day biotechnology. The present study was concerned with the production and partial characterization of extracellular amylase from Bacillus amyloliquefaciens P-001. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Enzyme production was induced by a variety of starchy substrate but corn flour was found to be a suitable natural source for maximum production. Tryptone and ammonium nitrate (0.2%) as nitrogen sources gave higher yield compared to other nitrogen sources. Maximum enzyme production was obtained after 48 hrs of incubation in a fermentation medium with initial pH 9.0 at 42°C under continuous agitation at 150 rpm. The size of inoculum was also optimized which was found to be 1% (v/v). Enzyme production was 2.43 times higher after optimizing the production conditions as compared to the basal media. Studies on crude amylase revealed that optimum pH, temperature and reaction time of enzyme activity was 6.5, 60°C and 40 minutes respectively. About 73% of the activity retained after heating the crude enzyme solution at 50°C for 30 min. The enzyme was activated by Ca2+ (relative activity 146.25%). It was strongly inhibited by Mn2+, Zn2+ and Cu2+, but less affected by Mg2+ and Fe2+.

Sulfide inhibition of and metabolism by cytochrome c oxidase.

Abstract: Hydrogen sulfide (H2S), a classic cytochrome c oxidase inhibitor, is also an in vitro oxidase substrate and an in vivo candidate hormonal (‘gasotransmitter’) species affecting sleep and hibernation. H2S, nitric oxide (NO) and carbon monoxide (CO) share some common features. All are low-molecular-mass physiological effectors and also oxidase inhibitors, capable of binding more than one enzyme site, and each is an oxidizable ‘substrate’. The oxidase oxidizes CO to CO2, NO to nitrite and sulfide to probable persulfide species. Mitochondrial cytochrome c oxidase in an aerobic steady state with ascorbate and cytochrome c is rapidly inhibited by sulfide in a biphasic manner. At least two successive inhibited species are involved, probably partially reduced. The oxidized enzyme, in the absence of turnover, occurs in at least two forms: the ‘pulsed’ and ‘resting’ states. The pulsed form reacts aerobically with sulfide to form two intermediates, ‘P’ and ‘F’, otherwise involved in the reaction of oxygen with reduced enzyme. Sulfide can directly reduce the oxygen-reactive a 3CuB binuclear centre in the pulsed state. The resting enzyme does not undergo such a step, but only a very slow one-electron reduction of the electron-transferring haem a . In final reactivation phases, both the steady-state inhibition of catalysis and the accumulation of P and F states are reversed by slow sulfide oxidation. A model for this complex reaction pattern is presented.

Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement

Abstract: Structural and functional studies reveal how the bacterial flavoenzyme EncM catalyses the oxygenation–dehydrogenation dual oxidation of a highly reactive substrate, and show that EncM maintains a stable flavin oxygenating species that promotes substrate oxidation and triggers a rarely seen Favorskii-type rearrangement. Flavoproteins, which contain either a flavin adenine dinucleotide or a flavin mononucleotide cofactor, are redox-active proteins involved in a broad range of biological processes including bioluminescence, photosynthesis and DNA repair. Here the authors undertook structural and functional studies to examine how the bacterial flavoenzyme EncM catalyses the oxygenation–dehydrogenation oxidation of a highly reactive substrate. They observed previously unknown flavin redox biochemistry: EncM maintains a stable flavin-oxygenating species that promotes substrate oxidation and triggers a rarely seen, Favorskii-type rearrangement that is central to the biosynthesis of the marine antibiotic enterocin. Flavoproteins catalyse a diversity of fundamental redox reactions and are one of the most studied enzyme families1,2. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate1,3,4. Here we report that the bacterial flavoenzyme EncM5,6 catalyses the peroxyflavin-independent oxygenation–dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics and isotope labelling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unexpected stable flavin-oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization.

Origins of activity enhancement in enzyme cascades on scaffolds.

Abstract: The concept of "metabolic channeling" as a result of rapid transfer of freely diffusing intermediate substrates between two enzymes on nanoscale scaffolds is examined using simulations and mathematical models. The increase in direct substrate transfer due to the proximity of the two enzymes provides an initial but temporary boost to the throughput of the cascade and loses importance as product molecules of enzyme 1 (substrate molecules of enzyme 2) accumulate in the surrounding container. The characteristic time scale at which this boost is significant is given by the ratio of container volume to the product of substrate diffusion constant and interenzyme distance and is on the order of milliseconds to seconds in some experimental systems. However, the attachment of a large number of enzyme pairs to a scaffold provides an increased number of local "targets", extending the characteristic time. If substrate molecules for enzyme 2 are sequestered by an alternative reaction in the container, a scaffold can result in a permanent boost to cascade throughput with a magnitude given by the ratio of the above-defined time scale to the lifetime of the substrate molecule in the container. Finally, a weak attractive interaction between substrate molecules and the scaffold creates a "virtual compartment" and substantially accelerates initial throughput. If intermediate substrates can diffuse freely, placing individual enzyme pairs on scaffolds is only beneficial in large cells, unconfined extracellular spaces or in systems with sequestering reactions.

Modifying Enzyme Activity and Selectivity by Immobilization

Abstract: Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.

Structure of the arginine methyltransferase PRMT5-MEP50 reveals a mechanism for substrate specificity

Abstract: The arginine methyltransferase PRMT5-MEP50 is required for embryogenesis and is misregulated in many cancers. PRMT5 targets a wide variety of substrates, including histone proteins involved in specifying an epigenetic code. However, the mechanism by which PRMT5 utilizes MEP50 to discriminate substrates and to specifically methylate target arginines is unclear. To test a model in which MEP50 is critical for substrate recognition and orientation, we determined the crystal structure of Xenopus laevis PRMT5-MEP50 complexed with S-adenosylhomocysteine (SAH). PRMT5-MEP50 forms an unusual tetramer of heterodimers with substantial surface negative charge. MEP50 is required for PRMT5-catalyzed histone H2A and H4 methyltransferase activity and binds substrates independently. The PRMT5 catalytic site is oriented towards the cross-dimer paired MEP50. Histone peptide arrays and solution assays demonstrate that PRMT5-MEP50 activity is inhibited by substrate phosphorylation and enhanced by substrate acetylation. Electron microscopy and reconstruction showed substrate centered on MEP50. These data support a mechanism in which MEP50 binds substrate and stimulates PRMT5 activity modulated by substrate post-translational modifications.

Transformation and removal of tetrabromobisphenol A from water in the presence of natural organic matter via laccase-catalyzed reactions: reaction rates, products, and pathways.

Abstract: The widespread occurrence of the brominated flame retardant tetrabromobisphenol A (TBBPA) makes it a possible source of concern. Our experiments suggest that TBBPA can be effectively transformed by the naturally occurring laccase enzyme from Trametes versicolor. These reactions follow second-order kinetics, whereby apparent removal rate is a function of both substrate and enzyme concentrations. For reactions at different initial concentrations and with or without natural organic matter (NOM), reaction products are identified using liquid or gas chromatography with mass spectrometry. Detailed reaction pathways are proposed. It is postulated that two TBBPA radicals resulting from a laccase-mediated reaction are coupled together via interaction of an oxygen atom on one radical and a propyl-substituted aromatic carbon atom on the other. A 2,6-dibromo-4-isopropylphenol carbocation is then eliminated from the radical dimer. All but one of the detected products arise from either substitution or proton elimination of the 2,6-dibromo-4-isopropylphenol carbocation. Three additional products are identified for reactions in the presence of NOM, which suggests that reaction occurs between NOM and TBBPA radical. Data from acute immobilization tests with Daphnia confirm that TBBPA toxicity is effectively eliminated by laccase-catalyzed TBBPA removal. These findings are useful for understanding laccase-mediated TBBPA reactions and could eventually lead to development of novel methods to control TBBPA contamination.

BN/CC Isosteric Compounds as Enzyme Inhibitors: N‐ and B‐Ethyl‐1,2‐azaborine Inhibit Ethylbenzene Hydroxylation as Nonconvertible Substrate Analogues

Abstract: BN/CC isosterism has recently emerged as a viable strategy to increase structural diversity.[1, 2] In particular, the chemistry of 1,2-dihydro-1,2-azaborines (hereafter abbreviated as 1,2-aza-borines), which are BN isosteres of the family of arenes, has attracted attention as novel aromatic compounds relevant to biomedical research and materials science. In the past decade, significant advances have been made in the synthesis and characterization of 1,2-azaborines.[3, 4] However, virtually no information is available on the behavior of 1,2-azaborines in a biological context. We previously demonstrated that N-ethyl-1,2-azaborine and the parent 1,2-azaborine bind inside the hydrophobic pocket of the L99A mutant of T4 lysozyme.[5] The demonstration of a biochemically active role of 1,2-azaborines, for example, as substrate or inhibitor of an enzyme, has to date remained elusive. In view of the dominance of arenes in pharmaceuticals, the study of the biochemical reactivity of 1,2-azaborines is significant: the new chemical space made available by 1,2-azaborines could open up opportunities in drug discovery and biomedical research. Herein, we reveal that the BN isosteres of ethylbenzene, N- and B-ethyl-1,2-azaborine, are actually strong inhibitors for the hydroxylation of ethylbenzene by ethylbenzene dehydrogenase (EbDH), providing the proof of concept that BN/CC isosterism can lead to novel biochemical behavior (Scheme 1). Ethylbenzene dehydrogenase is a molybdenum enzyme catalyzing the oxygen-independent hydroxylation of ethylbenzene to (S)-1-phenylethanol, initiating anaerobic ethylbenzene mineralization in the denitrifying betaproteo-bacterium “Aromatoleum aromaticum”.[6–10] Oxygen-independent hydroxylation of a non-activated hydrocarbon is an unusual and unique feature of this enzyme.[8] A potential reaction mechanism involving consecutive radical and carbocation derivatives of the substrates as reaction intermediates was derived from quantum chemical calculations and experimental data.[11–16] The enzyme hydroxylates an extraordinarily broad spectrum of alkylated aromatic and heteroaromatic compounds in an enantioselective manner,[12] making it a very suitable system to investigate 1,2-azaborines as potential substrates. Scheme 1 Effect of BN/CC isosterism on enzyme reactivity. Herein, we explore N-ethyl-1,2-azaborine and B-ethyl-1,2-azaborine as substrates for EbDH (Scheme 1). Somewhat surprisingly, we did not detect enzymatic turnover for either of the 1,2-azaborines by photometric assay or by GC-MS analysis of liquid-phase extractions of reactions incubated for up to 12 h. However, we were able to identify both N-ethyl-1,2-azaborine and B-ethyl-1,2-azaborine as very strong inhibitors of EbDH (see Figure 1a). In particular, N-ethyl-1,2-azaborine turned out to be a very efficient inhibitor with an IC50 value of 2.8 μm whereas B-ethyl-1,2-azaborine had an IC50 value of 100 μm. The nature of inhibition with N-ethyl-1,2-azaborine was investigated by a more detailed inhibitory kinetic analysis (Figure 1b). We determined N-ethyl-1,2-azaborine to be a mixed-type inhibitor with a very low competitive inhibition constant (Kic = 0.55 μm), which is basically identical to the Km of ethylbenzene (0.45 μm).[11] The uncompetitive inhibition constant (Kiu = 9 μm) is 16-fold higher than that value, suggesting the presence of a second binding site. The experimental observations are consistent with the very similar molecular/geometric structures between N-ethyl-1,2-azaborine and the native substrate ethylbenzene, which should make it hard for the enzyme to distinguish the two compounds. Figure 1 A: Nonlinear plots of the remaining ethylbenzene-dependent activity of EbDH when preincubated with different concentrations of N-ethyl-1,2-azaborine (circles) and B-ethyl-1,2-azaborine (squares). B: Double reciprocal plot of ethylbenzene-dependent enzymatic ... We further performed a MM (molecular mechanics) modeling (see Supporting Information) of the 1,2-azaborines inside the EbDH substrate binding pocket and of bound ethylbenzene to probe this hypothesis (Figure 2 and Supporting Information). Indeed, both 1,2-azaborines fit nicely in the pocket. In contrast to N-ethyl-1,2-azaborine, which is almost congruent with ethylbenzene, B-ethyl-1,2-azaborine seems to bind in a slightly different orientation to the active site. As can be seen from Figure 1a, B-ethyl-1,2-azaborine is a less-efficient inhibitor than N-ethyl-1,2-azaborine. Our modeling suggests that the lower inhibitory effect of B-ethyl-1,2-azaborine in comparison to N-ethyl-1,2-azaborine is a result of weaker binding through less-favorable van der Waals interactions with the hydrophobic residues in the active site (see Supporting Information for details). Figure 2 Overlay of N-ethyl-1,2-azaborine (red), B-ethyl-1,2-azaborine (yellow), and ethylbenzene (gray) modeled into the EbDH active site. The protein surface is given in grades of hydrophobicity (brown= hydrophobic, blue=hydrophilic). The view leads from the ... All the known substrates of EbDH are alkylaromatic compounds substituted at the C atoms of the aromatic ring.[11,12] To demonstrate that the relatively strong inhibitory behaviors of 1,2-azaborines originate from the combination of geometrical similarity and electronic structure differences between ethylbenzene and the corresponding 1,2-azaborines (BN/CC isosterism) rather than simple electronic structure changes through alkyl substitution at a heteroatom, we investigated the reactivity of EbDH with N-ethylimidazole and N-ethylpyrrole as other heteroatom-substituted hetero-cyclic substrate analogues. In doing so, we found that N-ethylimidazole is another inhibitory compound rather than a substrate, showing albeit a very weak inhibitory effect (IC50 value of 2200 μm) compared to N- and B-ethyl-1,2-azaborine. On the other hand, we detected clear enzymatic activity with N-ethylpyrrole, although it had a threefold lower rate than ethylbenzene (kcat: 0.16 s−1 and 0.4 s−1, respectively) and showed a relatively low affinity to EbDH (Km(app) value: 130 μm). The hydroxylation of N-ethylpyrrole proceeded with an observed enantiomeric excess (ee) of 60% for the S-enantiomer of the product 1-(1 H-pyrrol-1-yl)ethanol, determined by chiral GC coupled with mass spectrometry (see Supporting Information). We also detected a dehydrogenated side product (1-vinyl-1 H-pyrrole), derived from a carbocation intermediate postulated to be generated by EbDH.[12] 1-Vinyl-1 H-pyrrole may react non-enzymatically with water, resulting in a racemic mixture of the 1-(1 H-pyrrol-1-yl)ethanol enantiomers, which would explain the relatively low stereoselectivity observed for N-ethylpyrrole hydroxylation. To correlate the enzyme kinetic parameters of substrate conversion and enzyme inhibition to the molecular and electronic properties of the heteroatom-substituted compounds, calculations of the stabilities of corresponding radical and carbocation intermediates relative to those of ethylbenzene were performed (Table 1).[14] The data for N-ethyl-pyrrole show that radical formation is slightly less favored than for ethylbenzene. However, carbocation formation is energetically more favorable than for ethylbenzene, consistent with the involvement of a carbocation intermediate in the oxidation of N-ethylpyrrole by EbDH. Because substrate activation by radical formation is believed to be the rate limiting step,[16] these values may also explain the lower enzymatic activity with N-ethylpyrrole than with ethylbenzene. For the inhibitory compounds including the 1,2-azabor-ines, both calculated energies for radical and carbocation formation are substantially higher than those for ethylbenzene (Table 1). Therefore, it appears plausible that the energy barriers for forming the radical and carbocation intermediates cannot be overcome with these compounds, leading to inhibition by formation of non-productive enzyme–ligand complexes. These new data are consistent with the model for the catalytic mechanism of EbDH and will help to refine and expand the quantitative structure–activity relationship (QSAR) analysis for EbDH.[11] Table 1 Relative calculated energy levels for radical and carbocation formation from different ethylbenzene analogues. In summary, this study provides the first evidence that non-natural 1,2-azaborine compounds can influence enzyme activity as structural mimics of aromatic compounds. The differences in electronic structures between ethylbenzene and its BN isosteres result in a role change from substrate to inhibitor in EbDH-mediated hydroxylation reactions. Control experiments with other heteroatom-substituted alkylarenes suggest that the combined effects of geometric similarity and electronic structure differences provided by BN/CC isosterism are responsible for the potent inhibition. The data reported herein demonstrate the proof-of-concept that BN/ CC isosterism can lead to novel behavior in a biological context. The extended chemical space made available by azaborines should provide chemists with new tools to explore structure–activity relationships in biomedical research and medicinal chemistry.