Range Of Substrate Concentrations Research Articles

Relating switching rates between normal and persister cells to substrate and antibiotic concentrations: a mathematical modelling approach supported by experiments.

SummaryWe developed and compared two mathematical models of variable phenotypic switching rates between normal and persister cells that depend on substrate concentration and antibiotic presence. They could be used to simulate the formation of persisters in environments with concentration gradients such as biofilms. Our models are extensions of a previous model of the dynamics of normal and persistent cell populations developed by Balaban et al. (2004, Science 305: 1622). We calibrated the models’ parameters with experimental killing curves obtained after ciprofloxacin treatment of samples regularly harvested from planktonic batch cultures of Klebsiella pneumoniae. Our switching models accurately reproduced the dynamics of normal and persistent populations in planktonic batch cultures and under antibiotic treatment. Results showed that the models are valid for a large range of substrate concentrations and for zero or high doses of antibiotics.

A new general method for simultaneous fitting of temperature and concentration dependence of reaction rates yields kinetic and thermodynamic parameters for HIV reverse transcriptase specificity

Recent studies have demonstrated the dominant role of induced fit in enzyme specificity of HIV reverse transcriptase and many other enzymes. However, relevant thermodynamic parameters are lacking, and equilibrium thermodynamic methods are of no avail because the key parameters can only be determined by kinetic measurement. By modifying KinTek Explorer software, we present a new general method for globally fitting data collected over a range of substrate concentrations and temperatures and apply it to HIV reverse transcriptase. Fluorescence stopped-flow methods were used to record the kinetics of enzyme conformational changes that monitor nucleotide binding and incorporation. The nucleotide concentration dependence was measured at temperatures ranging from 5 to 37 °C, and the raw data were fit globally to derive a single set of rate constants at 37 °C and a set of activation enthalpy terms to account for the kinetics at all other temperatures. This comprehensive analysis afforded thermodynamic parameters for nucleotide binding (Kd , ΔG, ΔH, and ΔS at 37 °C) and kinetic parameters for enzyme conformational changes and chemistry (rate constants and activation enthalpy). Comparisons between wild-type enzyme and a mutant resistant to nucleoside analogs used to treat HIV infections reveal that the ground state binding is weaker and the activation enthalpy for the conformational change step is significantly larger for the mutant. Further studies to explore the structural underpinnings of the observed thermodynamics and kinetics of the conformational change step may help to design better analogs to treat HIV infections and other diseases. Our new method is generally applicable to enzyme and chemical kinetics.

Thread-based microfluidic chips as a platform to assess acetylcholinesterase activity.

In this paper, a microfluidic thread-based analytical device (μTAD) to assess the activity of acetylcholinesterase (AChE) via colorimetric analylsis is described. Fabrication of the device consists of two platforms, both with a nylon thread trifurcated into three channels terminating at open analysis sites at the end of the thread. 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) was spotted and dried on the analysis sites. Acetylthiocholine iodide (ATC) (or cysteine, Cys) is transported through an inlet channel of the nylon thread by capillary action due to the hydrophilic nature of nylon. AChE is transported through the other inlet channel and mixes with the ATC (or Cys) as they travel up to the analysis sites. As the solution reaches the analysis sites, an intense yellow color change occurs indicating the reaction of the thiol with DTNB to produce the yellow anion TNB2- . The sites are then dried, scanned, yielding a linear range of inverse yellow mean intensity versus substrate concentration. An IC50 value (1.74 nM) with a known inhibitor, neostigmine bromide (NB), is obtained on the device. The multiplex design enables triplicate data collection in a device that is easy to use. μTADs have great potential to be employed in a myriad of tests including point-of-care diagnostic devices for resource-challenged settings.

A microfluidic paper‐based device to assess acetylcholinesterase activity

Neurotransmitters play key roles in cell-to-cell communication. These chemical messengers are involved in many functional processes, including growth, reproduction, memory, and behavior. In this communication, we describe a novel microfluidic paper-based analytical device (μPAD) to detect acetylcholinesterase (AChE) activity and inhibitor screening through a colorimetric analysis. The μPAD is easily fabricated via a wax printing process whereby wax is deposited onto the surface of chromatographic paper, and heated to create a hydrophobic barrier. Separate solutions of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and samples containing AChE and acetylthiocholine iodide (ATC) (or cysteine, Cys), respectively, are directly spotted onto the μPAD. DTNB and AChE/ATC (or Cys) flow towards each other where a reaction occurs to form the yellow colored 2-nitro-5-thiobenzoic acid anion (TNB2- ). The device is dried, scanned, and analyzed yielding a linear range of average inverse yellow intensities versus substrate concentration. An IC50 value (0.045 nM) with a known inhibitor, neostigmine bromide (NB), is obtained on the device. μPADs are low cost and easy to fabricate and have great potential to quantify neurotransmitter activity.

PRODUCTION AND PARTIAL CHARACTERIZATION OF FIBRINOLYTIC ENZYME FROM A SOIL ISOLATE ASPERGILLUS CARBONARIUS S-CSR-0007

<p><strong>Objective: </strong>This work was undertaken with the aim of isolating and screening fungal soil isolates with fibrinolytic activity.</p><p><strong>Methods: </strong>Soil sample near slaughter house was collected and screened for fibrinolytic activity by using fibrin-agar. Enzyme production was optimized under various parameters like pH, temperature, substrate concentration and purified partially by ammonium sulphate precipitation. The stability of the partially purified enzyme was analyzed under the influence of a wide range of pH, temperature, and substrate concentrations.<strong></strong></p><p><strong>Results: </strong>Among the seven isolates screened, <em>Aspergillus carbonarius</em> S-CSR-0007 exhibited largest clear zone and was selected for further studies. Among the various substrates tested casein was found to support the highest caseinolytic activity of 816 U/ml and fibrinolytic activity of 510 U/ml. The culture supernatant of <em>A. carbonarius</em> S-CSR-0007 was fractionated by ammonium sulfate precipitation followed by dialysis, and maximum activity was obtained in the fraction with 80% ammonium sulfate, with an enzyme activity of 1200 U/ml using tyrosine as standard. The partially purified fibrinolytic enzyme showed optimal activity at 45 °C and pH 7.0. The enzyme was stable up to a temperature of 50 °C and pH 8.0, and the optimum substrate concentration was 4%.</p><p><strong>Conclusion: </strong>The crude enzyme showed high blood clot lysis activity, which may be a good candidate in the pharmaceutical industry. However, more studies need to be carried out to establish its clinical use.</p>

Enhancing the Enzymatic Activity of a Heme-Dependent Peroxidase through Genetic Modification

A heme-dependent peroxidase (HDP) catalyzes the ortho-hydroxylation of l-tyrosine to l-3,4-dihydroxyphenylalanine (l-DOPA) in the presence of hydrogen peroxide. l-DOPA can be used for the treatment of Parkinson's disease. In this work, to improve the catalytic efficiency, the heme-dependent peroxidase has been genetically modified with an elastin-like polypeptide (ELP). bicinchoninic acid (BCA) assay demonstrated that HDP-ELP has a higher solubility in aqueous solutions than HDP. Circular dichroism (CD) spectra showed that HDP-ELP has a higher stability than HDP. Enzyme kinetics has been investigated over a range of substrate concentrations. It has been demonstrated that HDP-ELP exhibited a catalytic efficiency 2.4 times that of HDP.

Cholinesterase inhibitory activity of isoquinoline alkaloids from three Cryptocarya species (Lauraceae)

Alzheimer’s disease is the most common form of dementia among older adults. Acetylcholinesterase and butyrylcholinesterase are two enzymes involved in the breaking down of the neurotransmitter acetylcholine. Inhibitors for these enzymes have potential to prolong the availability of acetylcholine. Hence, the search for such inhibitors especially from natural products is needed in developing potential drugs for Alzheimer’s disease. The present study investigates the cholinesterase inhibitory activity of compounds isolated from three Cryptocarya species towards acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Nine alkaloids were isolated; (+)-nornantenine 1, (−)-desmethylsecoantofine 2, (+)-oridine 3, (+)-laurotetanine 4 from the leaves of Cryptocarya densiflora BI., atherosperminine 5, (+)-N-methylisococlaurine 6, (+)-N-methyllaurotetanine 7 from the bark of Cryptocarya infectoria Miq., 2-methoxyatherosperminine 8 and (+)-reticuline 9 from the bark of Cryptocarya griffithiana Wight. In general, most of the alkaloids showed higher inhibition towards BChE as compared to AChE. The phenanthrene type alkaloid; 2-methoxyatherosperminine 8, exhibited the most potent inhibition against BChE with IC50 value of 3.95μM. Analysis of the Lineweaver–Burk (LB) plot of BChE activity over a range of substrate concentration suggested that 2-methoxyatherosperminine 8 exhibited mixed-mode inhibition with an inhibition constant (Ki) of 6.72μM. Molecular docking studies revealed that 2-methoxyatherosperminine 8 docked well at the choline binding site and catalytic triad of hBChE (butyrylcholinesterase from Homo sapiens); hydrogen bonding with Tyr 128 and His 438 residues respectively.

Towards a continuous two-phase partitioning bioreactor for xenobiotic removal

The removal of a xenobiotic (4-chlorophenol) from contaminated water was investigated in a simulated continuous two-phase partitioning bioreactor (C-TPPB), fitted with coiled tubing comprised of a specifically-selected extruded polymer, Hytrel 8206. Wastewater flowed inside the tubing, the pollutant diffused through the tubing wall, and was removed in the aqueous bioreactor phase at typical biological removal rates in the C-TTPB simulated by varying aqueous phase throughput to the reactor. Operating over a range of influent substrate concentrations (500–1500mgL−1) and hydraulic retention times in the tubing (4–8h), overall mass transfer coefficients were 1.7–3.5×10−7ms−1, with the highest value corresponding to the highest tubing flow rate. Corresponding mass transfer rates are of the same order as biological removal rates, and thus do not limit the removal process. The C-TPPB showed good performance over all organic and hydraulic loading ranges, with removal efficiencies of 4CP in the tubing wastewater stream always ≥96%. Additionally, the presence of the Hytrel tubing was able to buffer increases in organic loading to the hybrid system, enhancing overall process stability. Biological testing of the C-TPPB confirmed the abiotic test results demonstrating even higher 4-chlorophenol removal efficiency (∼99%) in the tubing stream.

Determination of kinetic parameters for casein hydrolysis by chymotrypsin using two ranges of substrate concentration

Hydrolysis of casein by chymotrypsin was performed at low and high substrate concentrations. Hydrolysis kinetics were analysed in the framework of a two-step model, with consecutive demasking and hydrolysis steps. This model took into consideration the limited accessibility of peptide bonds for the enzyme (masking effect). Dependence of the logarithm of the hydrolysis rate via degree of hydrolysis was non-linear at concentrations of 0.04 and 10 g L−1. In the intermediate range of substrate concentrations (0.07–0.72), this dependence was found to be linear, corresponding to the exponential model of proteolysis. Taking into consideration the multiplicity of peptide bonds, coupled to peptide bond demasking, allowed determination of two important parameters: the ratio of the rate constants of demasking and hydrolysis (kd/kh), and the fraction of initially masked bonds (m). An analytical procedure was suggested for the calculation of the apparent Michaelis constants at various degrees of hydrolysis.

Conformational rigidity of cholinesterases allows for the prediction of combined effects in a particular double mutant

The conformational rigidity of Drosophila melanogaster AChE, was checked by kinetic means on recombinant enzyme with the substitutions of two important amino acids, one at the catalytic anionic site (W83A), one at the peripheral anionic site (W321A) and the double mutant with both tryptophans substituted by alanines (W83A/W321A). It was hypothesized that the individual mutations would affect only the binding affinities of substrate molecules at each site and that a predictable effect would show up in the corresponding double mutant. Simple inspection revealed that bell shaped curves of activity at wide substrate concentration range in the catalytic anionic site mutants carry much less information than the analogous asymmetric ones of the wild type and peripheral anionic site mutant. Therefore, a concurrent kinetic analysis of the curves for all four enzymes was undertaken by constraining mutation independent parameters: unchanged affinity at the catalytic/peripheral anionic site of the opposite mutant in comparison to the parameters for wild type enzyme. Additionally, the parameters for W83A mutated enzyme were employed for the characterization of double mutant (W83A/W321A) protein by setting the dissociation constant for the substrate at the peripheral anionic site as determined for W321A mutant. Simultaneous analysis exactly reproduced the behavior of the double mutant without any significant change of previously reported values for the wild type enzyme (Stojan et al., 2004). This kinetic behavior is completely in line with the crystallographic evidence of structural rigidity in cholinesterases.

Electricity Production in Microbial Fuel Cell Subjected to Different Operational Modes

The effects of inoculum species, substrate concentration, temperature, and cathodic electron acceptors on electricity production of microbial fuel cells (MFCs) were investigated in terms of start-up time and power output. When inoculated with aeration tank sludge, this MFC outperformed the cell that was inoculated with anaerobic sludge in terms of start-up time and power output. After running for a certain time period, the dominant populations of the two MFCs varied significantly. Within the tested range of substrate concentration (200–1800 mg L−1), the voltage output increased and the time span of the electricity generation lengthened with increasing substrate concentration. As the temperature declined from 35 to 10 °C, the maximum power density reduced from 2.229 to 1.620 W m−3, and anodic polarization resistance correspondingly dropped from 118 to 98 Ω. The voltage output of MFC–Cu2+ was 0.447 V, which is slightly lower than that achieved with MFC–[Fe(CN)6]3− (0.492 V), thereby indicating that MFCs could be used to treat wastewater containing Cu2+ pollutant in the cathode chamber with removal of organics in anode chamber and simultaneous electricity generation.

Kinetics of the enzymatic hydrolysis of lignocellulosic materials at different concentrations of the substrate

The kinetics of the enzymatic hydrolysis of two substrates—lignocellulosic materials from Miscanthus and oat hulls—in an acetate buffer is studied at different concentrations of the substrates. The substrates are obtained via single-step treatment with a dilute solution of nitric acid. The content of a nonhydrolyzable component—acid-insoluble lignin—for Miscanthus and oat hulls was 11 and 14%, respectively. A multi-enzyme composition of commercially available enzyme preparations CelloLux-A and BrewZyme BGX was used as a catalyst. It is shown that treatment with the nitric acid solution produces reactive substrates for the enzymatic hydrolysis. The innovative science of the results is confirmed by Russian patent 2533921. Kinetics of the enzymatic hydrolysis of these substrates in an acetate buffer can be described by a mathematical model based on a modified Michaelis–Menten equation. The main kinetic constants for both substrates are determined from the experimental data. The equilibrium concentrations of reducing substances (RSes) for the substrates are calculated from the initial substrate concentrations. It is found that within the studied range of substrate concentrations (33.3–120.0 g/L), the initial rate of enzymatic hydrolysis for the lignocellulosic material from oat hulls is higher than that for the lignocellulosic material from Miscanthus by 1 g/(L h). It is shown that the yield of RS depends of the initial concentration of the substrates: as the concentration rises from 33.3 to 120 g/L, the yield of RS falls 1.5–2.0 times, due to substrate inhibition. At low initial concentrations, the yields of RS are similar for the substrates from Miscanthus and oat hulls. When the initial concentration of the substrate reaches 120 g/L, the yield of reducing substances for the lignocellulosic material from Miscanthus is approximately 20% higher than that for the lignocellulosic material from oat hulls. The established dependences and the proposed mathematical model allow us to optimize the initial concentration of the substrate for efficient enzymatic hydrolysis.

Fructose 1, 6 Bisphosphate Aldolase from Gestational Diabetic Human Placenta: Purification, Identification, and Investigation of Kinetic Properties

Gestational diabetes mellitus is described as glucose intolerance at various degrees that is first detected during pregnancy. In diabetic complications, there are changes in placental function, particularly with respect to intake, transmit, and utilization of glucose, and also in glycolysis and glycolytic enzymes. The placenta possibly plays a critical role in protecting the fetus from adverse effects caused by the maternal diabetic conditions. Fructose 1,6-bisphosphate aldolase, a main glycolytic enzyme, catalyses the cleavage of fructose 1,6-bisphosphate, resulting in two three-carbon products in many cells. In this study, we principally have investigated the presence of aldolase in diabetic human placenta and then purified the enzyme. We also determined the optimum conditions of enzyme assay measurements. With this procedure, we determined the specific activity of placental aldolase as 590, 94 mU/mg protein, and aldolase was purified about 63,0 fold from gestational diabetic human placenta. The molecular weight of human placental aldolase was found as 160 kDa. In present study, substrate kinetics were also investigated, and two different Km and Vmax values at high and low concentrations of substrate Fructose 1,6-bisphosphate were observed. High substrate concentration range is determined as the linear substrate concentration zone. Therefore, advanced kinetic studies had been performed at this linear zone. Enzymatic assays were carried out, and substrate kinetic properties were determined. According to this determination, Vmax value of gestational diabetic placental fructose 1,6-bisphosphate aldolase was found as 939,548±60,869 U/mg and Km as 24,304±2,948 mM.

Modeling Growth of Filaments and Floc Formers in Activated Sludge Flocs: Integrating the Effects of Kinetics and Diffusion

Filamentous bulking is the most common solid-separation problem in wastewater treatment systems. The phenomenon is attributed to competition between filamentous bacteria and floc-forming bacteria, a tradeoff between kinetic selection (KST) and substrate diffusion limitation (SDL). Previously, we explained our contradictory experimental results by both effects and developed a conceptual framework with three regions (bulking, intermediate, and non-bulking). However, within this purely qualitative framework, the range of substrate concentrations for each region was not determined. To test the framework and quantify the critical concentrations for the three regions, we here integrate KST and SDL into a model that simulates the growth rates of filaments and floc formers inside activated sludge flocs. Bulking and non-bulking sludges were produced in laboratory-scale reactors, and the kinetic model parameters were measured by respirometry. We then extended the model to verify our previous experimental results from sequencing batch reactors (SBRs). The effects of oxygen limitation and decay rates of floc formers and filaments on the modeled growth rates were also evaluated. In basic simulations, the model successfully determined the critical values of substrate concentrations in the framework. The results of the extended model were consistent with our experimental data, indicating that large flocs induce bulking whereas small flocs do not. Simulation results were more sensitive to the kinetic parameters than to the diffusion parameters, and the parameters more sensitively affected the growth of floc formers than filament growth.

Hide Dehairing and Laundry Detergent Compatibility Testing of Thermostable and Solvents Tolerant Alkaline Protease from Hot Spring Isolate <i>Bacillus cohnii</i>U3

In the present study, alkaline protease producing thermophilic Bacillus cohniiU3 strain was isolation from Unnai hot spring, India. The maximum production of protease (344 U/mL) was reported after 72 h of inoculation at 50°C in shake flask culture using the gelatin casein medium. The protease was found to remain active up to 96 h and production was growth dependent. The activity of parital purified protease was reported in abroad range of pH ranging from 4.0 to 11.0 with an optimum of 9.0 pH; Enzymes was indicated the highest activity at 50°C temperature. Salt independent catalysis and activity with a broad range of substrate concentration is the key feature of the protease. Thermos table nature, the stability in alkaline pH and stability in high salt concentration for 45 min were outstanding features of protease. The enzyme was stable in the presence of various organic solvents like Dimethyl Sulphoxide (DMSO), Methanol, Butanol, n-Hexane, Benzene at 25% (v/v) concentration. A good compatibility of the enzyme with most commercial detergents indicated its application in detergent industry. The remarkable dehairing in goat hide and destaining of blood spot in 2 h using 10 U/mL of protease assure that it could be a potential candidate for leather and detergent industries.

A model of the transient kinetics of laccase-catalyzed oxidation of phenol at micromolar concentrations

Oxidoreductase enzymes have the potential to be used to catalyze the oxidation of aromatic trace contaminants in wastewaters. The feasibility of this approach can be explored by modeling transient kinetics, particularly in the low substrate concentration range, where the model may be used to predict the quantity of enzyme and time required to achieve a particular level of conversion of a target compound. Laccase from Trametes versicolor was selected as a candidate enzyme due to its wide substrate specificity and its use of molecular oxygen as an oxidant. Phenol was selected as a target substrate. A four-parameter kinetic model was developed based on the known reactions of laccase. The model was applied to 30 sets of data collected from batch reactions conducted at pH 5 and 25°C over a three hour period. Initial phenol concentrations ranged from 0.5 to 50μM and applied enzyme concentrations ranged from 0.12 to 2.5μM. The model demonstrated its utility for predicting the quantities of enzyme and reaction times required to achieve desired levels of oxidation of phenol for varying initial concentrations. A three-parameter simplified version of the kinetic model was also developed to facilitate calibration and mathematical solution of its equations.

Characteristics of regulation of the activity of glucokinase from rat liver

A simple method for isolation of glucokinase from a soluble fraction of rat liver was proposed, making it possible to obtain the enzyme preparation characterized by high specific activity, native cooperative characteristics of the enzyme, and actually complete absence of other molecular forms of hexokinase. Dependence of the enzyme activity on the glucose concentration shows a pronounced sigmoidal behavior: with in the whole range of substrate concentrations at 25 and 2°C, the Hill coefficients are equal to 1.76 and 1.35, correspondingly, while the S0.5 values of glucose are 6.90 and 20 mM, respectively. The role of the kinetic reaction mechanism in the regulation of glucokinase activity in liver is under discussion.

Determination of the Catalytic Mechanism for Mitochondrial Malate Dehydrogenase

The kinetics of malate dehydrogenase (MDH) catalyzed oxidation/reduction of L-malate/oxaloacetate is pH-dependent due to the proton generated/taken up during the reaction. Previous kinetic studies on the mitochondrial MDH did not yield a consensus kinetic model that explains both substrate and pH dependency of the initial velocity. In this study, we propose, to our knowledge, a new kinetic mechanism to explain kinetic data acquired over a range of pH and substrate concentrations. Progress curves in the forward and reverse reaction directions were obtained under a variety of reactant concentrations to identify associated kinetic parameters. Experiments were conducted at physiologically relevant ionic strength of 0.17 M, pH ranging between 6.5 and 9.0, and at 25°C. The developed model was built on the prior observation of proton uptake upon binding of NADH to MDH, and that the MDH-catalyzed oxidation of NADH may follow an ordered bi-bi mechanism with NADH/NAD binding to the enzyme first, followed by the binding of oxaloacetate/L-malate. This basic mechanism was expanded to account for additional ionic states to explain the pH dependency of the kinetic behavior, resulting in what we believe to be the first kinetic model explaining both substrate and pH dependency of the reaction velocity.

Glutathione reductase a unique enzyme: molecular cloning, expression and biochemical characterization from the stress adapted C4 plant, Pennisetum glaucum (L.) R. Br.

The generation of excess reactive oxygen species (ROS) is one of the most common consequences of abiotic stress on plants. Glutathione reductase (GR, E.C. 1.6.4.2) and allied enzymes of the ascorbate-glutathione cycle play a crucial role to maintain the homeostatic redox balance in the cellular environment. GR plays an essential role in upholding the reduced glutathione pool under stress conditions. In the present study, a full-length GR cDNA and corresponding genomic clone was isolated from Pennisetum glaucum (L.) R. Br. The PgGR cDNA, encodes a 497-amino acid peptide with an estimated molecular mass of ~53.5 kDa. The PgGR peptide exhibits 54-89% sequence homology with GR from other plants and is cytoplasmic in nature. The PgGR enzyme was purified to near homogeneity, the recombinant protein being relatively thermostable and displaying activity in a broad range of temperature, pH and substrate concentrations. The PgGR transcript level was differentially regulated by heat, cold, salinity and methyl viologen-induced oxidative stress. The heterologously expressed PgGR protein in E. coli showed an improved protection against metal- and methyl viologen-induced oxidative stress. Our overall finding underscores the role of PgGR gene that responds to multiple abiotic stresses and provides stress tolerance in the experimental model (E. coli) which can be potentially used for the improvement of crops under abiotic stress conditions.

Flavin-Functionalized Gold Nanoparticles as an Efficient Catalyst for Aerobic Organic Transformations

Monolayer-protected gold clusters functionalized with synthetic flavins were synthesized and their catalytic activity in aerobic organic transformations investigated. Gold nanoparticles with 5-ethyl-3-(8-thiooctyl)lumiflavinium perchlorate acts as an efficient catalyst for the aerobic oxidation of organic sulfides to the corresponding sulfoxides upon treatment with hydrazine at room temperature and under atmospheric pressure in oxygen. With a catalytic amount of gold nanoparticles with 3-(8-thiooctyl)lumiflavin, diimide reduction of various olefins can be performed with hydrazine at room temperature under atmospheric pressure in air with greater yields of product alkanes than with non-supported 3-methyllumiflavin catalyst under the same conditions. Kinetic studies revealed that the monolayer-protected gold cluster-catalyzed reactions proceeded faster than those with non-supported catalysts over the full substrate concentration range for the hydrogenation of olefins and at lower substrate concentrations for sulfoxidation. This positive effect was rationalized by assuming a Michaelis–Menten-type mechanism in which the specific inclusion of substrates into the enzyme-like reaction cavities was a key factor in the high efficiency of the supported flavin catalysts.