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The intracellular environment displays complex dynamics influenced by factors such as molecular crowding and the low Reynolds number of the cytoplasm. Enzymes exhibiting active matter properties further heighten this complexity which can lead to memory effects. Molecular simulations often neglect these factors, treating the environment as a "thermal bath" using the Langevin equation (LE) with white noise. One way to consider these factors is by using colored noise instead within the generalized Langevin equation (GLE) framework, which allows for the incorporation of memory effects that have been observed in experimental data. We investigated the structural and dynamic differences in Shikimate kinase (SK) using LE and GLE simulations. Our results suggest that GLE simulations, which reveal significant changes, could be utilized for assessing conformational motions' impact on catalytic reactions. [ABSTRACT FROM AUTHOR]

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants—containing either substitution D395N or substitutions D395N+E456N—that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications. [ABSTRACT FROM AUTHOR]

The H 2 O 2 -assisted oxidation by a peroxidase enzyme takes place to help plants maintain the concentrations of organic compounds at physiological levels. Cells regulate the oxidation rate by inhibiting the action of this enzyme. The cells use two inhibitory processes to regulate the enzyme: a noncompetitive substrate inhibitory process and a competitive substrate inhibitory process. Numerous applications of peroxidase have been developed in clinical biochemistry, enzyme immunoassays, the treatment of waste water containing phenolic compounds, the synthesis of various aromatic chemicals, and the removal of peroxide from industrial wastes. The kinetic mechanism of the Spanish broom peroxidase enzyme is a Ping Pong Bi Bi mechanism with the presence of competitive inhibition by substrates. A mathematical model may help in identifying the key mechanism from amongst a set of competing mechanisms. In this study, we developed a fractional mathematical model to describe the H 2 O 2 -supported oxidation by the enzyme Spanish broom peroxidase. Numerical simulations of the model produced results that are consistent with the known behaviour of Spanish broom peroxidase. Finally, some future investigations of the study are briefly indicated as well. [ABSTRACT FROM AUTHOR]

The study presents a promising approach to enzymatic kinetics using Electrochemical Impedance Spectroscopy (EIS) to assess fundamental parameters of modified enteropeptidases. Traditional methods for determining these parameters, while effective, often lack versatility and convenience, especially under varying environmental conditions. The use of EIS provides a novel approach that overcomes these limitations. The enteropeptidase underwent genetic modification through the introduction of single amino acid modifications to assess their effect on enzyme kinetics. However, according to the one-sample t-test results, the difference between the engineered enzymes and hEKL was not statistically significant by conventional criteria. The kinetic parameters were analyzed using fluorescence spectroscopy and EIS, which was found to be an effective tool for the real-time measurement of enzyme kinetics. The results obtained through EIS were not significantly different from those obtained through traditional fluorescence spectroscopy methods (p value >> 0.05). The study validates the use of EIS for measuring enzyme kinetics and provides insight into the effects of specific amino acid changes on enteropeptidase function. These findings have potential applications in biotechnology and biochemical research, suggesting a new method for rapidly assessing enzymatic activity. [ABSTRACT FROM AUTHOR]

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon−oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis−Menten kinetic parameters for silicatein. [ABSTRACT FROM AUTHOR]

Enzyme research is important for the development of various scientific fields such as medicine and biotechnology. Enzyme databases facilitate this research by providing a wide range of information relevant to research planning and data analysis. Over the years, various databases that cover different aspects of enzyme biology (e.g., kinetic parameters, enzyme occurrence, and reaction mechanisms) have been developed. Most of the databases are curated manually, which improves reliability of the information; however, such curation cannot keep pace with the exponential growth in published data. Lack of data standardization is another obstacle for data extraction and analysis. Improving machine readability of databases is especially important in the light of recent advances in deep learning algorithms that require big training datasets. This review provides information regarding the current state of enzyme databases, especially in relation to the ever-increasing amount of generated research data and recent advancements in artificial intelligence algorithms. Furthermore, it describes several enzyme databases, providing the reader with necessary information for their use. [ABSTRACT FROM AUTHOR]

Featured Application: This manuscript describes the microwave inactivation kinetics of α-amylase enzyme for application as a time-temperature integrator in conventional or microwave heating. The demonstrated pH-dependent inactivation kinetics of α-amylase are valuable because they show that the thermal resistance of α-amylase varies with pH, and, therefore, a suitable pH can be used to employ α-amylase as a model for testing the efficiency of different thermal processes. The inactivation kinetics of an α-amylase enzymatic time-temperature integrator (TTI) from Bacillus subtilis (BAA) under continuous-flow microwave (MW) and conventional heating conditions were evaluated and compared in this study. The TTI dispersed in a buffer solution (pH 5.0 to 6.9) at 20 °C initially, and it was continuously circulated through two helical coils connected in a series for heating. The two coils were positioned in two domestic microwave ovens (2450 MHz and 1000 W nominal capacity each) and connected by a short tube. The sample flow rates were adjusted to result in a specific exit temperature in the range of 65 to 80 °C. A short fully insulated helical coil at the exit of the second oven was used as a holding tube. Test samples were drawn either at the exit of the second MW oven or immediately after the holding tube. The decimal reduction times obtained under conventional batch heating conditions decreased from 66 to 24 s as the temperature changed from 70 to 75 °C at pH 5.0 while they decreased from 8 to 5 s under MW in the same temperature range, but at pH 6.0, they increased both under conventional and microwave heating conditions (138 to 120 s and 89 to 61 s, respectively). The D-values under conventional thermal holding were four–eight times higher than under a continuous-flow MW heating condition. By varying the pH, the D-values could be modified to suit the validation of appropriate processing conditions. [ABSTRACT FROM AUTHOR]

Mimicking the active site and the substrate binding cavity of the enzyme to achieve specificity in catalytic reactions is an essential challenge. Herein, porous coordination cages (PCCs) with intrinsic cavities and tunable metal centers have proved the regulation of reactive oxygen species (ROS) generating pathways as evidenced by multiple photo‐induced oxidations. Remarkably, in the presence of the Zn4‐μ4‐O center, PCC converted dioxygen molecules from triplet to singlet excitons, whereas the Ni4‐μ4‐O center promoted the efficient dissociation of electrons and holes to conduct electron transfer towards substrates. Accordingly, the distinct ROS generation behavior of PCC‐6‐Zn and PCC‐6‐Ni enables the conversion of O2 to 1O2 and O2⋅−, respectively. In contrast, the Co4‐μ4‐O center combined the 1O2 and O2⋅− together to generate carbonyl radicals, which in turn reacted with the oxygen molecules. Harnessing the three oxygen activation pathways, PCC‐6‐M (M=Zn/Ni/Co) display specific catalytic activities in thioanisole oxidation (PCC‐6‐Zn), benzylamine coupling (PCC‐6‐Ni), and aldehyde autoxidation (PCC‐6‐Co). This work not only provides fundamental insights into the regulation of ROS generation by a supramolecular catalyst but also demonstrates a rare example of achieving reaction specificity through mimicking natural enzymes by PCCs. [ABSTRACT FROM AUTHOR]

Enzyme activation remains a largely under-represented and poorly exploited area of drug discovery despite some key literature examples of the successful application of enzyme activators by various mechanisms and their importance in a wide range of therapeutic interventions. Here we describe the background nomenclature, present the current position of this field of drug discovery and discuss the challenges of hit identification for enzyme activation, as well as our perspectives on the approaches needed to overcome these challenges in early drug discovery.

The interest in high hydrostatic pressure (HHP) is mostly focused on the inactivation of deleterious enzymes, considering the quality-related issues associated with enzymes in foods. However, more recently, HHP has been increasingly studied for several biotechnological applications, including the possibility of carrying out enzyme-catalyzed reactions under high pressure. This review aims to comprehensively present and discuss the effects of HHP on the kinetic catalytic action of enzymes and the equilibrium of the reaction when enzymatic reactions take place under pressure. Each enzyme can respond differently to high pressure, mainly depending on the pressure range and temperature applied. In some cases, the enzymatic reaction remains significantly active at high pressure and temperature, while at ambient pressure it is already inactivated or possesses minor activity. Furthermore, the effect of temperature and pressure on the enzymatic activity indicated a faster decrease in activity when elevated pressure is applied. For most cases, the product concentration at equilibrium under pressure increased; however, in some cases, hydrolysis was preferred over synthesis when pressure increased. The compiled evidence of the effect of high pressure on enzymatic activity indicates that pressure is an effective reaction parameter and that its application for enzyme catalysis is promising. [ABSTRACT FROM AUTHOR]

Extremophilic microorganisms, which are resistant to extreme levels of temperature, salinity, pH, etc., have become popular tools for biotechnological applications. Due to their availability and cost-efficacy, enzymes from extremophiles are getting the attention of researchers and industries in the field of biocatalysis to catalyze diverse chemical reactions in a selective and sustainable manner. In this mini-review, we discuss the advantages of Halomonas elongata as moderate halophilic bacteria to provide suitable enzymes for biotechnology. While enzymes from H. elongata are more resistant to the presence of salt compared to their mesophilic counterparts, they are also easier to produce in heterologous hosts compared with more extremophilic microorganisms. Herein, a set of different enzymes (hydrolases, transferases, and oxidoreductases) from H. elongata are showcased, highlighting their interesting properties as more efficient and sustainable biocatalysts. With this, we aim to improve the visibility of halotolerant enzymes and their uncommon properties to integrate biocatalysis in industrial set-ups. Keypoints: • Production and use of halotolerant enzymes can be easier than strong halophilic ones. • Enzymes from halotolerant organisms are robust catalysts under harsh conditions. • Halomonas elongata has shown a broad enzyme toolbox with biotechnology applications. [ABSTRACT FROM AUTHOR]

Glucose oxidase is an enzyme that is widely used in biosensors, especially kits for measuring blood sugar. Many diabetics use this type of kit to determine their blood sugar level. Aspergillus niger is the most important source of glucose oxidase for use in biosensors. Diabetes causes secondary diseases in patients for which medications are prescribed to improve them. Dexamethasone, a corticosteroid, is one of the drugs prescribed to diabetics to cure some secondary diseases. In this study, the effect of this drug on glucose oxidase was investigated from a kinetic and molecular point of view. In this study, the kinetics of drug binding to the enzyme was measured and the type of inhibition was determined by Lineweaver-Burk plot. The Ki value of the drug was determined by drawing the secondary curve. Using fluorescence spectrophotometry and molecular docking, the binding of the drug to the enzyme was confirmed. The results showed that the drug inhibits the enzyme non-competitively. Determining the kinetics parameters of the drug-enzyme interaction showed that the drug acts as a potent inhibitor. Study at the molecular level by fluorescence spectrophotometer showed that the drug attachment alters the enzyme conformation to more compaction. In silico results showed that the drug is placed between two helices that are outside the active site and binds to the enzyme by three hydrogen bonds. The result of this study is useful because it suggests that in diabetic patients taking dexamethasone, the amount of glucose declared by the kit may not be real due to the inhibition of glucose oxidase. [ABSTRACT FROM AUTHOR]

Chondroitinase ABC I (cABC I) from Proteus vulgaris is an important enzyme in medicinal biotechnology due to its ability to help axon regeneration after spinal cord injury. Its practical application involves solving several problems at the molecular and cellular levels. Structurally, most residues at the C-terminal domain of cABC I are arranged as organized strands, and only a small fraction of residues have helical conformation. The structural and functional features of modified residues on two specific helix fragments have previously been reported. The single mutant M889K has been combined with L679S and L679D mutants to make enzyme variants containing simultaneously modified helix. Here, the pH stability and temperature-based analysis of the transition state structure for the catalysis reaction were investigated. We found that double mutant L679D/M889K is the better choice to use in physiological conditions due to its higher pH stability at physiological pH as well as its different optimum temperature as compared with the (wild-type) WT protein. According to Arrhenius's analysis, the values of the Gibbs free energy of the transition state (∆G#) are not changed upon mutation. However, the relative contribution and absolute values of the enthalpy and entropy change to the total value of ∆G#, varied between the WT and mutants. [ABSTRACT FROM AUTHOR]

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is responsible for the conversion of atmospheric CO2 to organic carbon during photosynthesis, and often acts as a rate limiting step in the later process. Screening the natural diversity of Rubisco kinetics is the main strategy used to find better Rubisco enzymes for crop engineering efforts. Here, we demonstrate the use of Gaussian processes (GPs), a family of Bayesian models, coupled with protein encoding schemes, for predicting Rubisco kinetics from Rubisco large subunit (RbcL) sequence data. GPs trained on published experimentally obtained Rubisco kinetic datasets were applied to over 9000 sequences encoding RbcL to predict Rubisco kinetic parameters. Notably, our predicted kinetic values were in agreement with known trends, e.g. higher carboxylation turnover rates (Kcat) for Rubisco enzymes from C4 or crassulacean acid metabolism (CAM) species, compared with those found in C3 species. This is the first study demonstrating machine learning approaches as a tool for screening and predicting Rubisco kinetics, which could be applied to other enzymes. [ABSTRACT FROM AUTHOR]

Liquid-liquid phase separation (LLPS) is recognized as a mechanism for regulation of enzymatic activity. Biochemical mechanisms include concentrating reactants to enhance reaction rates or sequester enzymes and reactants from each other to reduce the reaction rate. On the other hand, LLPS might also regulate the diffusion of small molecules or important parameters for enzymatic activity (such as modulators, macromolecular crowding and changing the media physicochemical features) increasing or decreasing the reaction rate of the enzymes. Furthermore, the co-compartmentalization of specific enzymes can favour or speed up specific metabolic fluxes. Here, we discuss how LLPS contributed to generate a new era for enzyme regulation and the new possible subtle regulation mechanisms still unexplored. [ABSTRACT FROM AUTHOR]

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In solid organs, cells of the same "type" can vary in their molecular phenotype. The basis of this state variation is being revealed by characterizing cell features including the expression pattern of mRNAs and the internal distribution of proteins. Here, the variability of metabolic state between cells is probed by enzyme activity profiling. We study individual cells of types that can be identified during the post‐mitotic phase of oogenesis in Xenopus laevis. Whole‐cell homogenates of isolated oocytes are used for kinetic analysis of enzymes, with a focus on the initial reaction rate. For each oocyte type studied, the activity signatures of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and malate dehydrogenase 1 (MDH1) vary more between the homogenates of single oocytes than between repeat samplings of control homogenates. Unexpectedly, the activity signatures of GAPDH and MDH1 strongly co‐vary between oocytes of each type and change in strength of correlation during oogenesis. Therefore, variability of the kinetic behavior of these housekeeping enzymes between "identical" cells is physiologically programmed. Based on these findings, we propose that single‐cell profiling of enzyme kinetics will improve understanding of how metabolic state heterogeneity is related to heterogeneity revealed by omics methods including proteomics, epigenomics, and metabolomics. [ABSTRACT FROM AUTHOR]

Glutathione S-transferase (GST) detoxifies a broad spectrum of xenobiotics, especially in chemotherapeutic drugs, endogenous molecules, and environmental pollutants. Since the enzyme metabolizes toxic compounds, it has been extensively studied in many living things including aquatic organisms. In the current study, the GST enzyme was purified from brown meagre (Sciaena umbra) muscle tissue for the first time. Then, kinetic parameters of the enzyme were determined as optimum ionic strength: 20 mM Tris/HCl, optimum pH: 7.0 (Tris/HCl), and optimum substrate concentration: 3.125 mM. Eventually, inhibitory effects of the heavy metals were evaluated. IC50 values of the tested metal ions were calculated to be 0.1112, 0.6113, 0.727, and 0.7774 mM for Cd2+, Fe3+, Ag+, and Cu2+, respectively. Our results show that these heavy metals inhibit GST at very low concentrations which could cause dangerous results for aquatic systems. [ABSTRACT FROM AUTHOR]

The available carbon (C) to phosphorus (P) ratio in the soil is regulated by extracellular hydrolases for C and P acquisition by microbes and plants. However, the stoichiometric relationship between acquiring C and P in paddy rhizosphere and bulk soils remains unclear. The objective was to explore the underlying mechanisms of C and P acquisition stoichiometry in rhizosphere and bulk soils in response to P fertilization and cellulose addition. Amendment with either cellulose or P separately caused a significant increase in the maximal velocity (Vmax) of C acquisition enzymes (β‐1,4‐glucosidase and β‐cellobiohydrolase) but decreased that of P acquisition enzymes (acid and alkaline phosphomonoesterases) in bulk soil. In contrast, lower Vmax values of C and P acquisition enzymes were observed in rhizosphere soil than in bulk soil. The co‐application of cellulose and P increased the Vmax of P acquisition enzymes in rhizosphere soil but decreased that of only alkaline phosphomonoesterase in bulk soil. Results show that P availability and labile‐C content co‐regulated the P/C acquisition ratio, and two inverse linear relationships were observed. Specifically, the P/C acquisition ratio was negatively related to both the dissolved organic C/Olsen‐P ratio and the microbial biomass C/P ratio in rhizosphere soil. However, the P/C acquisition ratio was positively related to both the dissolved organic C/Olsen‐P ratio and the microbial biomass C/P ratio in bulk soil. Overall, microbes mineralized less organic P to acquire P in paddy soil rhizosphere (i.e., containing higher labile‐C) than in bulk soil (i.e., having lower labile‐C contents). [ABSTRACT FROM AUTHOR]

Introduction: Industrially, the use of enzymes has increased over time, as their production processes, stabilization and operating conditions have been optimized. Meanwhile associated with their use are minimized with environmental sustainability. The use of mathematical modeling has been a key factor in this advance, making it an great scientific interest area. Objective: To know the state of the art in this area of knowledge, highlighting the most successful and promising findings at present. Materials and Methods: A systematic search of recent high-impact publications between 2015 and 2021 was carried out in internationally recognized databases. A qualitative/comparative analysis of the approaches and contributions of the selected literature was carried out. Results and Discussion: It was evidenced that most of the mathematical models was developed from the Michaelis-Menten kinetic equation, but the simplifications assumed, the operating conditions and the mathematical methods used to solve these systems define the singularity and complexity of each investigation, which makes complex their comparison or implementation in a standardized way. Conclusions: In all cases, the proposed models were adjusted to experimental results or they agreed when different mathematical resolution methods were used. These results can provide valuable information for processes improvement and for equipment design, minimizing costs and time of experimentation and being more friendly to the environment. [ABSTRACT FROM AUTHOR]

Enzymes are be defined as soluble, colloidal, organic catalysts which are produced by living cells. Enzymes are biologic catalysts to accelerate the rate of biochemical reactions. in this study Quantification of the Enzyme Activity, Where: E = enzyme concentration; S = substrate concentration; ES = enzyme-substrate complex concentration; k1, k3= 1st order reaction rate constants (t-1); P = product concentration; k2= 2nd reaction rate constant (M-1.t-1). At first, it must be stressed that the formation of ES (enzyme-substrate complex) is an obligatory step to any type of enzyme-catalyzed reaction. The ES, depending on the reaction conditions, can form the product (forward reaction) or not (reward reaction). The immobilization technique allowed a more frequent use of biomaterials in industry, therapeutics (confection of subcutaneous capsules for controlled delivering of hormones), and lab analysis (automatic dosing equipments, enzyme electrode, biosensors and enzyme immunoassays). In industry, the use of glucose isomerase for the conversion of glucose into fructose (high fructose corn syrup), the aminoacylase for the separation of racemic mixture of amino acids, microbial lipase in triglyceride hydrolysis, and the lactase for removing lactose from milk and/or whey are stressed. [ABSTRACT FROM AUTHOR]

Beta-galactosidase (β-Gal) is an important enzyme utilized to determine cell senescence and understand disease pathology, among being utilized for several biosensing applications. This key biomarker can be measured indirectly using electrochemical methods by detecting the electroactive p-aminophenol (PAP) produced from the enzymatic conversion of a substrate. Here, we present a quick electrochemical method based on cyclic voltammograms (CVs) to quantify the amount of β-Gal in aqueous solution in under 5 minutes. Using timed CVs, the enzyme kinetics of β-Gal versus 4-aminophenyl-β-D-galactopyranoside (PAPG) was estimated. The detection limit for β-Gal was 40 ng ml−1 using the timed quick CV method. KM was found to be 18.7 ± 0.7 µM, and Vmax 10.1 ± 0.6 µmol min−1 mg−1, for β-Gal with PAPG as a substrate. By adding divalent salts Mg2+ and Ca2+, KM was lowered to nearly half, and Vmax increased by 20%. The electrochemical protocol utilized neutral pH and ambient room temperature benchtop conditions, making it amenable to scanning electrochemical microscopy and lab-on-chip detection platforms. In addition to cell senescence, this quick and simple electrochemical method can be used in broad biosensing applications, including the detection of water and foodborne pathogens and the identification of toxic contaminants. [ABSTRACT FROM AUTHOR]

The role of evolutionary pressure on the chemical step catalyzed by enzymes is somewhat enigmatic, in part because chemistry is not rate-limiting for many optimized systems. Herein, we present studies that examine various aspects of the evolutionary relationship between protein dynamics and the chemical step in two paradigmatic enzyme families, dihydrofolate reductases and alcohol dehydrogenases. Molecular details of both convergent and divergent evolution are beginning to emerge. The findings suggest that protein dynamics across an entire enzyme can play a role in adaptation to differing physiological conditions. The growing tool kit of kinetics, kinetic isotope effects, molecular biology, biophysics, and bioinformatics provides means to link evolutionary changes in structure-dynamics function to the vibrational and conformational states of each protein.

This investigation was carried out to study the characterization and kinetics of the partially purified Aspergillusficuumtannase enzyme for commercial scale application in the clarification and detannification of juices like grape. pH, temperature and optimum substrate concentration were determined for the tannase enzyme activity from Aspergillusficuum. Also, the Crude and partial purified enzyme were tested for detannification grape juice quality characteristics. Results evidenced that it was found that the optimum partial purified tannase parameters were: temperature 50 oC, PH 5.5 and substrate concentration 1.6% as a tannic acidwith 6.155 specific activities of partial purified tannase enzyme with 225.46% relative activity higher than its control. The tannase enzyme showed a higher temperature and lower pH optima than the soluble enzyme and showed 81.61% and 66.36 % tannin removal from grape juice by crude enzyme and partial purified enzyme treatment, respectively at 37 oC, which has not been reported earlier. Also, maximum velocity of reaction (Vmax) and Michaeli's constant (Km) values were 0.72 and 1.45 for partially purified tannase enzyme activity, respectively. Overall the quality of grape juice was better in partial pure tannase treated juice compared with untreated and crude enzyme treated juice. The present study exposed thatthis enzyme could demonstrate a valuable tool for a number of biotechnological applications. Also, the application of this enzyme on juice clarification also states that it can be employed in fruit juice processing for removal of tannin efficiently. [ABSTRACT FROM AUTHOR]

N‐alkylisonitrile, a precursor to isonitrile‐containing lipopeptides, is biosynthesized by decarboxylation‐assisted ‐N≡C group (isonitrile) formation by using N‐alkylglycine as the substrate. This reaction is catalyzed by iron(II) and 2‐oxoglutarate (Fe/2OG) dependent enzymes. Distinct from typical oxygenation or halogenation reactions catalyzed by this class of enzymes, installation of the isonitrile group represents a novel reaction type for Fe/2OG enzymes that involves a four‐electron oxidative process. Reported here is a plausible mechanism of three Fe/2OG enzymes, Sav607, ScoE and SfaA, which catalyze isonitrile formation. The X‐ray structures of iron‐loaded ScoE in complex with its substrate and the intermediate, along with biochemical and biophysical data reveal that ‐N≡C bond formation involves two cycles of Fe/2OG enzyme catalysis. The reaction starts with an FeIV‐oxo‐catalyzed hydroxylation. It is likely followed by decarboxylation‐assisted desaturation to complete isonitrile installation. [ABSTRACT FROM AUTHOR]

Aminopeptidase N is an important metalloenzyme from the M1 zinc metallopeptidase family, which is present in numerous apicomplexan parasites, including Plasmodium, Eimeria, and Cryptosporidium. Aminopeptidase N is a potential drug target, and hence, its properties have been widely investigated. In the current study, the cellular localization and enzyme characteristics of Toxoplasma gondii aminopeptidase N3 (TgAPN3) were evaluated in vitro. Cellular localization analysis revealed that TgAPN3 and GRA protein were co-located in the organelle and parasitophorous vacuole of T. gondii. The secretion assay showed that TgAPN3 could be co-secreted from the tachyzoites with GRA protein. A functional recombinant Toxoplasma aminopeptidase N3 (rTgAPN3) was produced in Escherichia coli. The enzyme activity was first determined using a fluorogenic H-Ala-MCA substrate. Some activity of rTgAPN3 was observed between pH 3.0 and 8.0, with a peak at pH 7.0. The activity was significantly enhanced in the presence of Co2+ ions. Substrate specificity of rTgAPN3 was then evaluated. The enzyme showed a preference for substrates containing N-terminal Ala residues, followed by Tyr and Cys. The rTgAPN3 activity was significantly inhibited by bestatin and phebestatin. In general, TgAPN3 was a structurally conserved member of the M1 family, although it also displayed unique biochemical characteristics. These results lay the foundation for a functional study of TgAPN3 and constitute its putative identification as a drug target. [ABSTRACT FROM AUTHOR]

Plasmin is a potent serin protease involved in a variety of biological functions, such as fibrinolysis and tissue remodeling. On performing an in vitro control assay to measure the activity of endogenous plasmin in cell lysates, a stimulatory effect of non-ionic detergent NP-40 on plasmin activity was discovered. Another non-ionic detergent, TX-100, also enhanced plasmin activity, while ionic detergents sodium deoxycholate and sodiem dodecyl sulfate abolished plasmin enzyme activity. Kinetic analysis of plasmin activity in the presence of NP-40 and TX-100 demonstrated an increase in V max ; however, there was no change in K m values, suggesting that these detergents stimulate plasmin activity in a non-competitive manner. Fibrin plate assay indicates that NP-40 and TX-100 functionally stimulate plasmin activity by showing a dose-dependent increase in fibrinolysis. • Plasmin activity is stimulated by nonionic detergents Nonidet P-40 and Triton X-100. • Nonidet P-40 and Triton X-100 facilitate plasmin enzyme kinetics in a non-competitive manner. • Nonidet P-40 and Triton X-100 increase fibrinolysis in fibrin plate assay by functionally stimulating plasmin activity. [ABSTRACT FROM AUTHOR]

Abstract Here, we evaluate spiroacridines as inhibitors of tyrosinase, a key enzyme to melanogenesis. For this purpose, the spiroacridines 3-(acridin-9-yl)-N-benzylidene-2-cyanoacrylohydrazide (AMTAC-01) and 3-(acridin-9-yl)-2-cyano-N-(4-metoxybenzylidene)-acrylohydrazide (AMTAC-02) were synthesized and their enzymatic inhibition types and mechanisms were investigated. In addition, the interaction of these compounds with the enzyme were studied by UV–Vis spectroscopy, spectrofluorimetry, 1H NMR titration as well as molecular docking. Spectroscopic results reveals that the acridine derivatives interact strongly (K a ≅ 104 − 105 M−1) with the mushroom tyrosinase and the enzyme undergoes small structural modifications due to the interaction with AMTAC-01 compound. The interaction studies support the enzymatic inhibition results, which suggests that AMTAC-01 compounds inhibit the enzyme reversibly and follows a noncompetitive type (AMTAC-01) and mixed type (AMTAC-02) of inhibition. Nevertheless, AMTAC-02 (IC 50 = 96.29 μM) inhibits the enzyme more effectively than AMTAC-01 (IC 50 = 189.40 μM), which suggests a highly relevant role of AMTAC-02's methoxy group to the inhibition activity, which is confirmed by docking studies to mushroom tyrosinase. Docking also indicates this interaction to be absent in human tyrosinase. Significance Based on previous results which evidenced the relevant activity of two spiroacridinic compounds for cell growth inhibition against melanoma cells, here we improve our understanding about the spiroacridines in the biological media by exploring the molecular mechanism that govern the activities of these two compounds using mushroom tyrosinase (mTYR) enzyme as molecular target. The paper not only will have a major impact upon molecular mechanism that regulates melanin inhibition by spiroacridinic compounds, but also by guiding the search for enzyme inhibitors and the development of new anti-melanoma prophylaxis. [ABSTRACT FROM AUTHOR]

A novel kinetic method was developed for the quantitation of α-ketoglutaric acid (AKG) in cardioplegic solution and athletic supplements. The assay relied on an enzymatic transamination of AKG and d -4-hydroxyphenylglycine to form 4-hydroxybenzoylformic acid and l -glutamic acid using d -phenylglycine aminotransferase. Since 4-hydroxybenzoylformic acid absorbed UV strongly at 334 nm, the initial rate of the reaction was determined by the increasing absorbance at this wavelength without the need for colorimetric probes or coupling reactions, and this information was used for the construction of a standard curve against AKG concentration. The method showed good linearity (r 2  = 0.9994) over an AKG concentration range of 20–160 μM. The limits of detection and quantitation were 4.09 and 13.62 μM respectively. It was simple, inexpensive, accurate and precise, as well as repeatable, and was not interfered with by excipients in the samples. Regarding the environmental friendliness, the method was free from the use of organic solvents or hazardous reagents and required no chemical pre-treatment of samples. The proposed method gave assay results tested in real samples in agreement with the HPLC method and commercial assay kits, therefore being suitable for routine analysis of AKG in quality control laboratories. [ABSTRACT FROM AUTHOR]

In this work, variation iteration method is used to develop analytical solutions for the prediction of molar concentration of native and denatured jack bean urease (EC 3.5.1.5) through the three-reaction steps kinetic model of thermal inactivation of the urease. The obtained analytical solutions are used to study the kinetics of thermal inactivation of the enzyme as applied in biotechnology. The analytical solutions are verified with numerical solutions using Runge-Kutta with shooting method and good agreements are established between the solutions. The information given in this theoretical investigation will assist in the kinetic analysis of the experimental results over handling rate constants and molar concentrations. The analytical solutions as developed in this work can serve as a starting point for a better understanding of the relationship between the physical quantities of the problems. [ABSTRACT FROM AUTHOR]

Brushes of vertically-standing enzyme-containing nanotubes are prepared onto planar surfaces by a combination of hard-templating and layer-by-layer assembly. The nanotubes have a core-shell morphology made of two compartments, one for mechanical rigidity, the other containing β -lactamase for bioactivity. We demonstrate inclusion of the enzymatic component either in the core or in the shell part of the nanotubes. Kinetic studies reveal that both types of systems are bioactive but that the activity is significantly better preserved over long time periods when β -lactamase is incorporated in the core of the nanotubes. [ABSTRACT FROM AUTHOR]

Natural and synthetic derivatives that contain an indole core are being used in medical treatments and technological processes. Therefore, the development of new synthetic methods for the synthesis of indole derivatives is very popular. In this study, new oxindoles with reaction of 4,7-dihydro-1H-indole (2) and isatin (4) were synthesized as analogs of natural product 3,3'-bis(indolyl)oxindole. The biological properties of the compounds obtained during this study were also studied, showing that compounds 5, 7, and 12 inhibited the activity of G6PD with an IC50 of 99 μM, 231 μM, and 304 μM respectively. The activity of rat erythrocyte 6PGD was increased in the presence of 5 and 7 and was inhibited in the presence of 12. As indole derivative 5 was an activator of 6PGD and inhibitor of G6PD, it was selected for docking studies to understand the mechanism of activation and inhibition. [ABSTRACT FROM AUTHOR]

Abstract: Method that could regulate the ion transport in nanochannel in an efficient and rapid manner is still a challenge. Here, we introduced enzyme‐catalysis‐induced polymer growth in nanochannels to develop a new method to regulate the ion transport and evaluate the enzyme catalysis kinetics in nano‐space. As a model enzyme, Horseradish peroxidase (HRP) was immobilized in the nanochannels through a volume‐controlled‐drying method. In the presence of H2O2, HRP catalyzed o‐phenylenediamine (o‐PD) to trigger its polymer growth, in turn blocked the ion transport and led to the decrease of the ion current. Taking advantages of the high efficiency of enzyme catalysis and the nano‐confinement of nanochannels, the system readily achieved blocking ratios of ion current even reaching 99.6 % of the initial. Based on above concept, we developed a new method to evaluate the enzyme catalysis kinetics in nano‐confined space. By comparing with those in free state in solution and absorbed on planar surface, HRP confined in nanochannels presented similar apparent Michaelis constant (Km) values for the substrate H2O2 but much higher Km values for the substrate o‐PD, due to the steric hindrance and diffusion suppression. The enzyme‐catalysis‐induced polymerization in nanochannels might lead to new concept for the nano‐blocking/switching and provide a new platform for single molecule analysis and detection. [ABSTRACT FROM AUTHOR]