Initial Reaction Velocity Research Articles

Estimation of the kinetic parameters (Km & Vmax) and the optimization of yeast alcohol dehydrogenase (ADH) assay.

Alcohol dehydrogenases (ADHs) are zinc-containing enzymes that catalyze the oxidation of alcohols to aldehydes or ketones. The enzymes also play a critical role in the metabolism of a number of drugs and metabolites containing alcohol functional groups. Kinetic parameters of yeast alcohol dehydrogenase (ADH) was estimated using spectrophotometer, optimum temperature and pH for ethanol, as well as ADH specificity to other substrates was determined. Results from the assay showed value for the initial velocity of reaction of the enzyme estimated as 0.297. Change in concentration of NADH which correspond to ADH activity for ethanol was calculated as 48nanokatals (4.8 X 10-5 mol-1l), the results also showed average values of the Km and Vmax of ADH for ethanol as estimated from Michaelis-Menten and Line weaver-Burk double reciprocal plots, to be 21.5mM and 0.426 respectively. Optimum temperature and pH of ADH for ethanol were estimated to be 25 °C and 8 respectively. Although, reaction specificity of ADH to other substrates showed gradation between propan-2-ol and ethanol with ethanol showing highest activity and methanol least. 2-propene-1-ol also presented high enzyme activity relative to ethanol. In most of enzyme assays, enzyme activity is determined by measuring the rate of conversion of substrate or rate of production of products within a given period of time. In this experiment, the rate of oxidation of NADH was monitored since NADH has a known maximum light absorbance at 340nm.

Determination of monophenolase activity based on backpropagation neural network analysis of three-dimensional fluorescence spectroscopy

Tyrosinase is pivotal for melanin formation. Measuring monophenolase activity is of great importance for both fundamental research and industrial applications. For the first time, a backpropagation (BP) artificial neural network with three-dimensional fluorescence spectroscopy was applied for the real-time determination of tyrosinase monophenolase activity. Principal component analysis (PCA) was utilized for the dimension reduction of three-dimensional fluorescence data. The four principal components served as inputs for the neural network. Network parameters were optimized using a genetic algorithm (GA). BP learning algorithm was applied to train the network model to determine tyrosine levels in a binary mixture containing tyrosine and L-DOPA without any chemical separation. The time course of tyrosine consumption by monophenolase was determined to calculate the initial velocity of the enzymatic reaction. The limit of detection of the monophenolase assay was 0.0615 U·mL−1. This combined strategy of PCA, GAs, and BP artificial neural networks for three-dimensional fluorescence spectroscopy was efficient for the real-time and in-situ determination of monophenolase activity in a cascade reaction.

A two-enzyme system in an amorphous metal-organic framework for the synthesis of D-phenyllactic acid.

In this study, we synthesized an amorphous metal-organic framework by adjusting the concentration of precursors, and established a two-enzyme system consisting of lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH), which successfully achieved coenzyme recycling, and applied it to the synthesis of D-phenyllactic acid (D-PLA). The prepared two-enzyme-MOF hybrid material was characterized using XRD, SEM/EDS, XPS, FT-IR, TGA, CLSM, etc. In addition, reaction kinetic studies indicated that the MOF-encapsulated two-enzyme system exhibited faster initial reaction velocities than free enzymes due to its amorphous ZIF-generated mesoporous structure. Furthermore, the pH stability and temperature stability of the biocatalyst were evaluated, and the results indicated a significant improvement compared to the free enzymes. Moreover, the amorphous structure of the mesopores still maintained the shielding effect and protected the enzyme structure from damage by proteinase K and organic solvents. Finally, the remaining activity of the biocatalyst for the synthesis of D-PLA reached 77% after 6 cycles of use, and the coenzyme regeneration still maintained at 63%, while the biocatalyst also retained 70% and 68% residual activity for the synthesis of D-PLA after 12 days of storage at 4 °C and 25 °C, respectively. This study provides a reference for the design of MOF-based multi-enzyme biocatalysts.

Fabrication of a Ratiometric Fluorescence Sensor Based on Carbon Dots as Both Luminophores and Nanozymes for the Sensitive Detection of Hydrogen Peroxide.

The construction of novel fluorescent nanozymes is highly desirable for providing new strategies for nanozyme-based sensing systems. Herein, a novel ratiometric fluorescence sensing platform was constructed based on carbon dots (CDs) as both luminophores and nanozymes, which could realize the sensitive detection of hydrogen peroxide (H2O2). CDs with peroxidase-mimicking activity were prepared with a one-step hydrothermal method using L-histidine as an inexpensive precursor. CDs had bright blue fluorescence. Due to the pseudo-peroxidase activity, CDs catalyzed the oxidation of o-phenylenediamine (OPD) with H2O2 to generate 2,3-diaminophenolazine (DAP). The fluorescence resonance energy transfer (FRET) between CDs and DAP resulted in a decrease in the fluorescence of CDs and an increase in the fluorescence of DAP, leading to a ratiometric fluorescence system. The free radical trapping experiment was used to investigate the reactive oxygen radicals (ROS) in the catalytic process of CD nanozymes. The enzymatic parameters of CD nanozymes, including the Michaelis constant (Km) and the maximum initial reaction velocities (Vmax), were investigated. A good affinity for both OPD and H2O2 substrates was proven. Based on the FRET between CDs and OPD, a ratiometric fluorescence analysis of H2O2 was achieved and results ranged from 1 to 20 μM and 20 to 200 μM with a low limit of detection (LOD, 0.42 μM). The detection of H2O2 in milk was also achieved.

Superior Peroxidase-Like Activity of Gold Nanorattles in Ultrasensitive H2 O2 Sensing and Antioxidant Screening.

Nanozymes are artificial enzyme systems which are easy to produce, highly stable and cost-effective in comparison to natural enzymes. Herein, we evaluated the peroxidase-like activity of gold nanorattles (Au NRTs) having a solid gold octahedron core and thin, porous cubic gold shell. We also prepared solid gold nanocubes and nanospheres of similar sizes and surface charge as that of Au NRTs and compared their activity with standard horse radish peroxidase (HRP). All the prepared nanostructures followed Michaelis-Menten kinetics as observed from their substrate concentration vs. initial reaction velocity plot using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The kinetic parameters and the catalytic efficiency for the peroxidase-like activity of the nanostructures and HRP were calculated, and it was observed that Au NRTs possess the best nanozymatic activity with lowest KM and highest catalytic efficiency (kcat /KM ). The better activity of Au NRTs compared with other nanostructures and HRP could be attributed to the hollow porous structure with a solid core where different surfaces are available for reaction. Au NRTs, being the best amongst the tested nanozymes were further used for the sensing of hydrogen peroxide (H2 O2 ) and were found to sense H2 O2 down to 0.5 μM. Further, two naturally occurring antioxidants, tannic acid and ascorbic acid showed inhibitory effect on the peroxidase-like activity of Au NRTs in a concentration dependent manner which can be further used for screening of antioxidants or for determining the antioxidant potential.

Molecular mechanisms and biochemical analysis of fluorene degradation by the Pseudomonas sp. SMT-1 strain.

Fluorene is a harmful organic toxicant extensively disseminated in the water and dry land ecosystem. Its toxicity and ubiquitous presence pose issues concerning its biodegradation. Characterization of the molecular mechanisms of fluorene degradation, detection of metabolites, and appraisal of its viability in toxicant removal by the SMT-1 Pseudomonas sp. strain are the main purposes of this study. In this work, the catabolic intermediates were identified from resting cell reactions of the SMT-1 strain as well as the involved catabolic pathway of fluorene. Based on liquid chromatography mass spectrometry analysis, the identified intermediates were 9-fluorenone; 3,4-dihydroxy-9-fluorenone; phthalate and protocatechuic acid. The specific primers were designed to amplify the fluorene-degrading 4921 dioxygenase gene segment from the SMT-1 Pseudomonas sp. strain. The 4921 dioxygenase gene was expressed, purified and characterized. The apparent K m and V max values were 25.99µMmin-1 and 0.77 U mg-1, respectively. The enzyme was most active at pH 7.5 and 25°C in Tris-HCl buffer and was identified by measuring the initial reaction velocity for 1min. Effect of metal salts on enzyme activity was accessed to see the impact on protein stability. Most of the analyzed metal salts inhibited enzyme activity to different degrees, and exhibited very low activity in the presence of FeCl3. Understanding the physiological, metabolic pathway and molecular mechanism of fluorene degradation is an important factor in increasing significant information of this biological process. This strain may serve as a potential candidate for further use in the bioremediation process to treat organic toxicant contaminated sites.

Essential moieties of myricetins, quercetins and catechins for binding and inhibitory activity against α-Glucosidase

α-Glucosidase inhibition of 11 flavonoids, including myricetins, quercetins and catechins were studied through initial reaction velocity, IC50 value, inhibition kinetics, fluorescence quenching and molecular docking. It was found that C4 = O, C2 = C3, 3-OH and 5′-OH were essential moieties for α-glucosidase inhibition of myricetin that was shown with the highest inhibitory activity. The trans-conformational catechins was shown with stronger inhibition effects than the cis-conformational ones. Further, gallocatechin was an uncompetitive inhibitor, while myricetin, myricetrin, quercetin and catechin were competitive ones. 3-OH and 5′-OH promoted myricetin to bind with the enzyme active site through hydrogen bondings. The presence of C4 = O and C2 = C3 increased electron delocalization in ring A-C for myricetin and quercetin, and this enhanced stability of π-conjugations with aromatic residues of amino acids. However, 5′-OH decreased the quenching effects because it limited π-conjugations of ring B with key fluorescent residues. Notably, for same flavonoid sort, the constants that indicate binding affinity of flavonoids to α-glucosidase, including reciprocal of competitive inhibition constant, fluorescence quenching constant and binding energy followed same order as the inhibitory activity, indicating that α-glucosidase inhibition of the flavonoids resulted from binding interactions between them, and that the methods above can be combined reasonably to characterize flavonoid-enzyme binding interactions.

Discriminating between Concerted and Sequential Allosteric Mechanisms by Comparing Equilibrium and Kinetic Hill Coefficients.

Hill coefficients, which provide a measure of cooperativity in ligand binding, can be determined for equilibrium (or steady-state) data by measuring fractional saturation (or initial reaction velocities) as a function of ligand concentration. Hill coefficients can also be determined for transient kinetic data from plots of the observed rate constant of the ligand-promoted conformational change as a function of ligand concentration. Here, it is shown that the ratio of the values of these two Hill coefficients can provide insight into the allosteric mechanism. Cases when the value of the kinetic Hill coefficient is equal to or greater than the value of the equilibrium coefficient indicate concerted transitions whereas ratios smaller than one indicate a sequential transition. The derivations in this work are for symmetric dimers but are expected to have general applicability for homo-oligomers.

Kinetics of Allosteric Inhibition of Single Enzyme by Product Molecules.

Single-molecule experiment probed the catalytic conversions of Amplex Red to resorufin by horseradish peroxidase in which the product molecules were found to act as the allosteric inhibitor for the individual enzyme. While broad distributions of the initial reaction velocities and the number of product molecules required to cease the reaction unraveled the underlying dynamic disorder in the reaction pathway, bulk experimental measurements established the particularity of noncompetitive inhibition of the enzyme species. In this work, to rationalize the observed phenomena, we present a stochastic kinetic model of the enzymatic reaction taking into account the inhibitions of the enzyme and enzyme-substrate complex in the circumstance of their structural fluctuations. Starting from a chemical master equation that can further be reduced into a set of ordinary differential equations for the case of single-enzyme kinetics, we derived an analytical expression for the turnover time distribution, the first moment of which yielded the mean turnover time. The inverse of the latter showed excellent agreement with the bulk data of the initial enzymatic velocities measured in the presence of varying inhibitor concentrations. This supports the observed nature of inhibition which we further confirmed by constructing double-reciprocal plots for the inhibited kinetics. In addition, we successfully recovered ensemble data from another experiment that redesigned the aforesaid single-molecule catalysis to investigate the effects of molecular crowders on reaction velocity; the latter was greatly alleviated with increasing the molecular weight of the crowders. On the other hand, the calculated randomness parameter, determined from the higher moment of the turnover time distribution, clearly inferred dynamic disorder in the catalytic turnovers. Our work under a unified framework provides a robust theoretical description for the experimental kinetic study and eradicates the necessity of assuming an alternative mode of inhibition in analyzing the data, not consistent with the experiments, considered earlier.

Optimization and validation of a fluorogenic dipeptidyl peptidase 4 enzymatic assay in human plasma

During the development of a specific dipeptidyl peptidase 4 (DPP4) inhibitor to treat type 2 diabetes, a fluorogenic kinetic analysis for DPP4 enzymatic activity using Gly-Pro-Aminomethylcoumarin (AMC) as a substrate was optimized and validated for recombinant DPP4 and human plasma samples. The sensitivity, calibration curve, detection range, accuracy, precision, recovery efficiency, Km constant, short/long-term stability, and stability after freezing-thawing cycles were analyzed. DPP4 enzymatic activity (mU/min) was measured as the initial velocity (Vo) of the enzymatic reaction over time. The sensitivity of the Vo value was 14,488 mU/min for recombinant DPP4 and 17,995 mU/min for human plasma samples. The dynamic ranges of the calibration curve were linear and reliable between 1.11 × 104–1.86 × 106 mU/min of the mean Vo value and in the DPP4 concentration range of 23.4–3,000 ng/mL. The assay's accuracy and precision met acceptance criteria for all samples. Plasma DPP4 was stable under various storage temperatures, even after three freeze-thaw cycles. Our optimized, validated bioanalytic method for measuring DPP4 activity in plasma samples was successfully employed to evaluate the effect of evogliptin (DA-1229) tartrate, which irreversibly and dose-dependently inhibits DPP4 enzymatic activity, without the dilution effect of human plasma samples and irrespective of the co-treated metformin.

Crowded environment affects the activity and inhibition of the NS3/4A protease

Kinetic parameters characterizing the catalytic activities of enzymes are typically investigated in dilute solutions. However, in reality, these reactions occur in cells that, in addition to water and ions, are full of other macromolecules including proteins, nucleic acids, lipids, and metabolites. Such a crowded environment might affect enzyme-catalyzed reaction rates, so it is necessary to mimic the crowd in laboratory settings.We determined the effect of macromolecular crowders on the activity of the hepatitis C virus protease NS3/4A. As crowders we used polyethylene glycol (PEG), Ficoll, and bovine serum albumin. Using the fluorescence assay with a labeled peptide substrate, we found that the crowders affected the kinetics of the NS3/4A-catalyzed reaction differently. The Ficoll crowders increased and PEG decreased the initial and maximum reaction velocities. To explain the opposite effects exerted by PEG as compared to Ficoll, we performed molecular dynamics simulations of NS3/4A in explicit solvent and surrounded by its peptide substrates and PEG molecules. The simulations suggest both hydrophobic and polar/electrostatic interactions between PEG and NS3/4A with hydrogen bonds formed between PEG oxygens and NS3/4A amino acids rich in hydrogen bonds donors. The NS3/4A protease is a known target for telaprevir, an anti-viral drug. We found that Ficoll changes the inhibition constant for telaprevir suggesting that the effect of crowders should also be considered in inhibitor design.

Relationship between enzyme concentration and Michaelis constant in enzyme assays

The upper bound of enzyme concentration for accurately estimating the parameters in Michaelis-Menten (MM) equation is not completely determined and still under discussion, even though many researchers have investigated the equation’s validity for a long time. In the paper, we broadly investigated the correlation between the system of ordinary differential equations for monosubstrate irreversible enzyme reaction (HMM-system) and its derivative MM equation focusing on the relationship between initial enzyme concentration [E]0 and Michaelis constant Km by numerical simulation. According to the results, the initial reaction velocity v0 is still a function of initial substrate concentration [S]0 at [E]0<Km. The function is identical to the MM equation at [E]0≦0.01Km, while it is described as a new type of equation at 0.01Km≦[E]0<Km. This function is of great significance in enzyme assays as a comprehensive approximation for the HMM-system.

Enzymatic Synthesis of Glucose Monodecanoate in a Hydrophobic Deep Eutectic Solvent.

Environmentally friendly and biodegradable reaction media are an important part of a sustainable glycolipid production in the transition to green chemistry. Deep eutectic solvents (DESs) are an ecofriendly alternative to organic solvents. So far, only hydrophilic DESs were considered for enzymatic glycolipid synthesis. In this study, a hydrophobic DES consisting of (-)-menthol and decanoic acid is presented for the first time as an alternative to hydrophilic DES. The yields in the newly introduced hydrophobic DES are significantly higher than in hydrophilic DESs. Different reaction parameters were investigated to optimize the synthesis further. Twenty milligrams per milliliter iCalB and 0.5 M glucose resulted in the highest initial reaction velocity for the esterification reaction, while the highest initial reaction velocity was achieved with 1.5 M glucose in the transesterification reaction. The enzyme was proven to be reusable for at least five cycles without significant loss of activity.

Optimization of Glycolipid Synthesis in Hydrophilic Deep Eutectic Solvents.

Glycolipids are considered an alternative to petrochemically based surfactants because they are non-toxic, biodegradable, and less harmful to the environment while having comparable surface-active properties. They can be produced chemically or enzymatically in organic solvents or in deep eutectic solvents (DES) from renewable resources. DES are non-flammable, non-volatile, biodegradable, and almost non-toxic. Unlike organic solvents, sugars are easily soluble in hydrophilic DES. However, DES are highly viscous systems and restricted mass transfer is likely to be a major limiting factor for their application. Limiting factors for glycolipid synthesis in DES are not generally well understood. Therefore, the influence of external mass transfer, fatty acid concentration, and distribution on initial reaction velocity in two hydrophilic DES (choline:urea and choline:glucose) was investigated. At agitation speeds of and higher than 60 rpm, the viscosity of both DES did not limit external mass transfer. Fatty acid concentration of 0.5 M resulted in highest initial reaction velocity while higher concentrations had negative effects. Fatty acid accessibility was identified as a limiting factor for glycolipid synthesis in hydrophilic DES. Mean droplet sizes of fatty acid-DES emulsions can be significantly decreased by ultrasonic pretreatment resulting in significantly increased initial reaction velocity and yield (from 0.15 ± 0.03 μmol glucose monodecanoate/g DES to 0.57 ± 0.03 μmol/g) in the choline: urea DES. The study clearly indicates that fatty acid accessibility is a limiting factor in enzymatic glycolipid synthesis in DES. Furthermore, it was shown that physical pretreatment of fatty acid-DES emulsions is mandatory to improve the availability of fatty acids.

An approach to better estimation of the intial velocity of enzyme reactions

A new algorithm for computation of initial velocity of enzyme reaction at the time zero is proposed. This algorithm makes it possible to reduce systematic error of measurements to the minimum, to estimate reaction velocity in testing samples regardless of the enzyme activity levels and to minimize assay time. The study is illustrated by an example of salivary alpha-amylase and standard reagent kit. The algorithm should not be applied if conjugated enzyme systems are used because there is a long initial lag-phase in the kinetic curve.

An Approach to Better Estimation of the Initial Velocity of Enzyme Reactions.

A new algorithm for computation of the initial velocity of enzyme reaction at the time zero is proposed. This algorithm makes it possible to reduce the systematic error of measurements to the minimum, to estimate the reaction velocity in test samples regardless of the enzyme activity levels, and to minimize the assay time. The study is illustrated by an example of salivary alpha-amylase and standard reagent kit. The algorithm should not be applied if conjugated enzyme systems are used because there is a long initial lag phase in the kinetic curve.

Real-Time Interferometric Refractive Index Change Measurement for the Direct Detection of Enzymatic Reactions and the Determination of Enzyme Kinetics.

Back scatter interferometry (BSI) is a sensitive method for detecting changes in the bulk refractive index of a solution in a microfluidic system. Here we demonstrate that BSI can be used to directly detect enzymatic reactions and, for the first time, derive kinetic parameters. While many methods in biomedical assays rely on detectable biproducts to produce a signal, direct detection is possible if the substrate or the product exert distinct differences in their specific refractive index so that the total refractive index changes during the enzymatic reaction. In this study, both the conversion of glucose to glucose-6-phosphate, catalyzed by hexokinase, and the conversion of adenosine-triphosphate to adenosine di-phosphate and mono-phosphate, catalyzed by apyrase, were monitored by BSI. When adding hexokinase to glucose solutions containing adenosine-triphosphate, the conversion can be directly followed by BSI, which shows the increasing refractive index and a final plateau corresponding to the particular concentration. From the initial reaction velocities, KM was found to be 0.33 mM using Michaelis–Menten kinetics. The experiments with apyrase indicate that the refractive index also depends on the presence of various ions that must be taken into account when using this technique. This study clearly demonstrates that measuring changes in the refractive index can be used for the direct determination of substrate concentrations and enzyme kinetics.

Comparison of deep eutectic solvents and solvent-free reaction conditions for aldol production

In this work, we investigated the catalytic aldolase activity of the porcine pancreas lipase (PPL) for 4-nitrobenzaldehyde (4-NBA) and acetone in different deep eutectic solvent (DES) compared with bovine serum albumin (BSA)-catalyzed aldol reaction. The PPL catalyzed the aldol addition specifically towards the desired aldol product, especially in hydrophobic DES. In contrast to this result, BSA-catalyzed aldol additions did not exhibit specificity for neither aldol nor olefin formation. For the PPL-catalyzed reactions, the product composition could be directly correlated with the choice of DES, differing in their hydrophobicity. The initial reaction velocity of the aldol addition was higher in the hydrophilic DES ChCl:Gly. However, this DES was limited by the solubility for the 4-NBA substrate. The 4-NBA containing DES exhibited the highest solubility for 4-NBA. However, the fastest reaction with a final yield of 1296.5 mM, which corresponds to 71% yield and 82% conversion after 32 h, was achieved in the co-solvent acetone, making this reaction solvent-less and improving the productivity up to 40.5 mM h−1 compared to reactions performed in DES.

FRET-Based Assays to Determine Calpain Activity.

Calpains are signaling proteases that have relatively little sequence specificity but some preferences for certain residues on either side of the scissile bond. As with most proteases, they mainly cut unstructured or extended regions of their target proteins. The tendency for concentrated calpain to rapidly autoproteolyze when activated by calcium complicates the kinetic assessment of calpain activity. As calpain autoproteolyzes, the amount of fully active enzyme continuously decreases until all of the calpain molecules have been cut and their activity reduced to a tiny fraction of the starting rate. To accurately measure calpain kinetics, only the initial rate of substrate hydrolysis, where autoproteolysis is minimal, can be used. To accomplish this, a method for rapid, quantifiable determination of substrate cleavage is required. Many of the existing assays are lacking in their sensitivity to accurately quantify calpain activity within this timeframe. However, the FRET peptide substrates developed by Cuerrier et al. have been shown to have sufficiently high affinity between substrate and enzyme to accurately measure the initial enzyme reaction velocity at substrate concentrations above the Km value. With a suitably sensitive fluorimeter, sufficient data can be obtained to evaluate calpain kinetics and inhibition. Here we describe a facile, reliable calpain assay based on the continuous monitoring of FRET fluorescence from the highly sensitive calpain-specific substrate, (EDANS)-EPLFAERK-(DABCYL). We illustrate some difficulties associated with determining kinetic constants of whole calpains that are simultaneously undergoing autoproteolysis and how the assay can be used to help characterize calpain-specific inhibitors. We also present a variation of this fluorescence-based assay for high-throughput screening using the calpain protease core and a fluorescence plate reader.

Improved octyl glucoside synthesis using immobilized β-glucosidase on PA-M with reduced glucose surplus inhibition

A β-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (V0) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219 mM and 116 mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336 h of reaction, the OG yield and the conversion rate of glucose reached 134 mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively.